HIV-1 drug-resistance patterns among patients on failing treatment in a large number of European countries

Background: Information about patterns of HIV-1 drug resistance among treatment-exposed patients is crucial for the development of novel effective drugs. Currently no system exists that monitors patterns of resistance in patients failing therapy. Methods: The study included 1,988 HIV-1 sequences from patients experiencing therapy failure collected between 2000 and 2004 in 15 European countries. Genotypic resistance was interpreted using the ANRS algorithm. Phenotypic resistance was predicted using the Virco geno- to phenotype system. Results: 80.7% of the sequences included at least one drug-resistance mutation. Mutations were found for NRTIs (73.5%), NNRTIs (48.5%), and protease inhibitors (35.8%). Ninety percent of sequences with genotypic resistance harbored M184V, M41L, K103N, D67N, and/or T215Y. Among NRTIs, resistance was most frequently predicted for lamivudine. About half of all sequences had reduced susceptibility for NNRTIs. Resistance to most boosted protease inhibitors was found in &lt; 25%. No sequence had resistance to all currently available drugs. Conclusion: Levels of resistance among patients with therapy failure were high. The patterns of resistance reflect resistance to drugs available for a longer time. Fully suppressive regimens can be designed even for the most mutated HIV because boosted protease inhibitors have remained active against most circulating viruses and new drug classes have become available.</p

Elevated risk of infection with SARS-CoV-2 Beta, Gamma, and Delta variants compared with Alpha variant in vaccinated individuals

The extent to which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) break through infection- or vaccine-induced immunity is not well understood. We analyzed 28,578 sequenced SARS-CoV-2 samples from individuals with known immune status obtained through national community testing in the Netherlands from March to August 2021. We found evidence of an increased risk of infection by the Beta (B.1.351), Gamma (P.1), or Delta (B.1.617.2) variants compared with the Alpha (B.1.1.7) variant after vaccination. No clear differences were found between vaccines. However, the effect was larger in the first 14 to 59 days after complete vaccination compared with ≥60 days. In contrast to vaccine-induced immunity, there was no increased risk for reinfection with Beta, Gamma, or Delta variants relative to the Alpha variant in individuals with infection-induced immunity.</p

Effect of HIV-1 low-level viraemia during antiretroviral therapy on treatment outcomes in WHO-guided South African treatment programmes : A multicentre cohort study

Background: Antiretroviral therapy (ART) that enables suppression of HIV replication has been successfully rolled out at large scale to HIV-positive patients in low-income and middle-income countries. WHO guidelines for these regions define failure of ART with a lenient threshold of viraemia (HIV RNA viral load ≥1000 copies per mL). We investigated the occurrence of detectable viraemia during ART below this threshold and its effect on treatment outcomes in a large South African cohort. Methods: In this observational cohort study, we included HIV-positive adults registered between Jan 1, 2007, and May 1, 2016, at 57 clinical sites in South Africa, who were receiving WHO-recommended ART regimens and viral load monitoring. Low-level viraemia was defined as the occurrence of at least one viral load measurement of 51-999 copies per mL during ART. Outcomes were WHO-defined virological failure (one or more viral load measurement of ≥1000 copies per mL) and switch to second-line ART. Risks were estimated with Cox proportional hazard models. Findings: 70 930 patients were included in the analysis, of whom 67 644 received first-line ART, 1476 received second-line ART, and 1810 received both. Median duration of follow-up was 124 weeks (IQR 56-221) for patients on first-line ART and 101 weeks (IQR 51-178) for patients on second-line ART. Low-level viraemia occurred in 16 013 (23%) of 69 454 patients, with an incidence of 11·5 per 100 person-years of follow-up (95% CI 11·4-11·7), during first-line ART. Virological failure during follow-up occurred in 14 380 (22%) of 69 454 patients on first-line ART. Low-level viraemia was associated with increased hazards of virological failure (hazard ratio [HR] 2·6, 95% CI 2·5-2·8; p<0·0001) and switch to second-line ART (HR 5·2, 4·4-6·1; p<0·0001]) compared with virological suppression of less than 50 copies per mL. Risk of virological failure increased further with higher ranges and persistence of low-level viraemia. Interpretation: In this large cohort, low-level viraemia occurred frequently and increased the risk of virological failure and switch to second-line ART. Strategies for management of low-level viraemia need to be incorporated into WHO guidelines to meet UNAIDS-defined targets aimed at halting the global HIV epidemic. Funding: None

Viral reactivations and associated outcomes in the context of immune reconstitution after pediatric hematopoietic cell transplantation

Background: Viral reactivations (VRs) after hematopoietic cell transplantation (HCT) contribute to significant morbidity and mortality. Timely immune reconstitution (IR) is suggested to prevent VR. Objectives: We studied the relation between IR (as a continuous predictor over time) and VR (as a time-varying predictor) and the relation between VR and other clinical outcomes. Methods: In this retrospective analysis all patients receiving a first HCT between January 2004 and September 2014 were included. IR (CD3/CD4/CD8 T, natural killer, and B cells) was measured biweekly until 12 weeks and monthly thereafter. Main outcomes of interest were VR of adenovirus, EBV, human herpesvirus 6 (HHV6), cytomegalovirus (CMV), and BK virus screened weekly. Clinical outcomes included overall survival (OS), event-free-survival, nonrelapse mortality (NRM), and graft-versus-host disease. Cox proportional hazard and Fine and Gray competing risk models were used. Results: Two hundred seventy-three patients (age, 0.1-22.7 years; median follow-up, 58 months) were included. Delayed CD4 reconstitution predicted reactivation of adenovirus (hazard ratio [HR], 0.995; P = .022), EBV (HR, 0.994; P = .029), and HHV6 (HR, 0.991; P = .012) but not CMV (P = .31) and BK virus (P = .27). Duration of adenovirus reactivation was shorter with timely CD4 reconstitution, which was defined as 50 × 106 cells/L or greater within 100 days. Adenovirus reactivation predicted lower OS (HR, 2.17; P = .0039) and higher NRM (HR, 2.96; P = .0008). Concomitant CD4 reconstitution abolished this negative effect of adenovirus reactivation (OS, P = .67; NRM, P = .64). EBV and HHV6 reactivations were predictors for the occurrence of graft-versus-host disease, whereas CMV and BK virus reactivation did not predict clinical outcomes. Conclusion: These results stress the importance of timely CD4 reconstitution. Strategies to improve CD4 reconstitution can improve HCT outcomes, including survival, and reduce the need for toxic antiviral therapies

Impact of the HIV-1 genetic background and HIV-1 population size on the evolution of raltegravir resistance

BACKGROUND: Emergence of resistance against integrase inhibitor raltegravir in human immunodeficiency virus type 1 (HIV-1) patients is generally associated with selection of one of three signature mutations: Y143C/R, Q148K/H/R or N155H, representing three distinct resistance pathways. The mechanisms that drive selection of a specific pathway are still poorly understood. We investigated the impact of the HIV-1 genetic background and population dynamics on the emergence of raltegravir resistance. Using deep sequencing we analyzed the integrase coding sequence (CDS) in longitudinal samples from five patients who initiated raltegravir plus optimized background therapy at viral loads > 5000 copies/ml. To investigate the role of the HIV-1 genetic background we created recombinant viruses containing the viral integrase coding region from pre-raltegravir samples from two patients in whom raltegravir resistance developed through different pathways. The in vitro selections performed with these recombinant viruses were designed to mimic natural population bottlenecks. RESULTS: Deep sequencing analysis of the viral integrase CDS revealed that the virological response to raltegravir containing therapy inversely correlated with the relative amount of unique sequence variants that emerged suggesting diversifying selection during drug pressure. In 4/5 patients multiple signature mutations representing different resistance pathways were observed. Interestingly, the resistant population can consist of a single resistant variant that completely dominates the population but also of multiple variants from different resistance pathways that coexist in the viral population. We also found evidence for increased diversification after stronger bottlenecks. In vitro selections with low viral titers, mimicking population bottlenecks, revealed that both recombinant viruses and HXB2 reference virus were able to select mutations from different resistance pathways, although typically only one resistance pathway emerged in each individual culture. CONCLUSIONS: The generation of a specific raltegravir resistant variant is not predisposed in the genetic background of the viral integrase CDS. Typically, in the early phases of therapy failure the sequence space is explored and multiple resistance pathways emerge and then compete for dominance which frequently results in a switch of the dominant population over time towards the fittest variant or even multiple variants of similar fitness that can coexist in the viral population

High rates of transmission of drug-resistant HIV in Aruba resulting in reduced susceptibility to the WHO recommended first-line regimen in nearly half of newly diagnosed HIV-infected patients

Background. In Western countries emergence of human immunodeficiency virus (HIV) drug resistance has tremendously decreased, and transmission of drug resistance has merely stabilized in recent years. However, in many endemic settings with limited resources rates of emerging and transmitted drug resistance are not regularly assessed. Methods. We performed a survey including all HIV-infected individuals who received resistance testing in 2010-2015 in Aruba, a highly endemic HIV area in the Caribbean. Transmitted HIV drug resistance was determined using World Health Organization (WHO) criteria. Transmission dynamics were investigated using phylogenetic analyses. In a subset, baseline samples were re-analyzed using next generation sequencing (NGS). Results. Baseline resistance testing was performed in 104 newly diagnosed untreated individuals (54% of all newly diagnosed individuals in 2010-2015): 86% were men, 39% were foreign-born, and 22% had AIDS at diagnosis. And 33% (95% CI: 24-42%) was infected with a drug-resistant HIV variant. The prevalence of resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) reached 45% (95% CI: 27-64%) in 2015, all based on the prevalence of mutation K103N. NGS did not demonstrate additional minority K103N-variants compared to routine resistance testing. K103N-harboring strains were introduced into the therapy-unexposed population via at least 6 independent transmissions epidemiologically linked to the surrounding countries. Virological failure of the WHO-recommended first-line NNRTI-based regimen was higher in the presence of K103N. Conclusions. The prevalence of resistant HIV in Aruba has increased to alarming levels, compromising the WHO-recommended first-line regimen. As adequate surveillance as advocated by the WHO is limited, the Caribbean region could face an unidentified rise of NNRTI-resistant HIV

Rapid Rebound of a Preexisting CXCR4-tropic Human Immunodeficiency Virus Variant After Allogeneic Transplantation With CCR5 Δ32 Homozygous Stem Cells

Allogeneic stem cell transplantation (alloSCT) of homozygous CCR5 δ32 stem cells once resulted in the cure of human immunodeficiency virus (HIV) infection. We have recently reported a viral breakthrough in a similar setting. Here, we demonstrate that the rapid rebound after alloSCT was related to a highly replicative CXCR4-tropic HIV variant, which could already be detected before alloSCT

All-printed cell counting chambers with on-chip sample preparation for point-of-care CD4 counting

We demonstrate the fabrication of fully printed microfluidic CD4 counting chips with complete on-chip sample preparation and their applicability as a CD4 counting assay using samples from healthy donors and HIV-infected patients. CD4 counting in low-income and resource-limited point-of-care settings is only practical and affordable, if disposable tests can be fabricated at very low cost and all manual sample preparation is avoided, while operation as well as quantification is fully automated and independent of the skills of the operator. Here, we show the successful use of (inkjet) printing methods both to fabricate microfluidic cell counting chambers with controlled heights, and to deposit hydrogel layers with embedded fluorophore-labeled antibodies for on-chip sample preparation and reagent storage. The maturation process of gelatin after deposition prevents antibody wash-off during blood inflow very well, while temperature-controlled dissolution of the matrix ensures complete antibody release for immunostaining after the inflow has stopped. The prevention of antibody wash-off together with the subsequent complete antibody release guarantees a homogeneous fluorescence background, making rapid and accurate CD4 counting possible. We show the successful application of our fully printed CD4 counting chips on samples from healthy donors as well as from HIV-infected patients and find an excellent agreement between results from our method and from the gold standard, flow cytometry, in both cases