Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir

HIV persists in latently infected cells of patients on antiretroviral therapy (ART). This persistent proviral DNA reservoir is an important predictor of viral rebound upon therapy failure or interruption and forms a major obstacle towards cure. Accurate quantification of the low levels of persisting HIV DNA may aid patient monitoring and cure research. Digital PCR is a promising tool that enables direct absolute quantification with high sensitivity. With recent technological advances, several platforms are available to implement digital PCR in a clinical setting. Here, we compared two digital PCR platforms, the Quantstudio 3D (Life Technologies) and the QX100 (Bio-Rad) with a semi-nested qPCR on serial HIV DNA dilutions and DNA isolated from PBMCs of ART-suppressed patients. All three methods were able to detect target to the lowest levels of 2.5 HIV DNA copies. The QX100 excelled in having the least bias and highest precision, efficiency and quantitative linearity. Patient sample quantifications by the QX100 and semi-nested qPCR were highly agreeable by Bland-Altman analysis (0.01 ± 0.32 log10). Due to the observation of false-positive signals with current digital PCR platforms however, semi-nested qPCR may still be preferred in a setup of low quantity detection to discriminate between presence or absence of HIV DNA

Real-life validation of the Panbio (TM) COVID-19 antigen rapid test (Abbott) in community-dwelling subjects with symptoms of potential SARS-CoV-2 infection

Background: RT-qPCR is the reference test for identification of active SARS-CoV-2 infection, but is associated with diagnostic delay. Antigen detection assays can generate results within 20 min and outside of laboratory settings. Yet, their diagnostic test performance in real life settings has not been determined. Methods: The diagnostic value of the Panbio™ COVID-19 Ag Rapid Test (Abbott), was determined in comparison to RT-qPCR (Seegene Allplex) in community-dwelling mildly symptomatic subjects in a medium (Utrecht, the Netherlands) and high endemic area (Aruba), using two concurrently obtained nasopharyngeal swabs. Findings: 1367 and 208 subjects were enrolled in Utrecht and Aruba, respectively. SARS-CoV-2 prevalence, based on RT-qPCR, was 10.2% (n = 139) and 30.3% (n = 63) in Utrecht and Aruba respectively. Specificity of the Panbio™ COVID-19 Ag Rapid Test was 100% (95%CI: 99.7–100%) in both settings. Test sensitivity was 72.6% (95%CI: 64.5–79.9%) in the Netherlands and 81.0% (95% CI: 69.0–89.8%) in Aruba. Probability of false negative results was associated with RT-qPCR Ct-values, but not with duration of symptoms. Restricting RT-qPCR test positivity to Ct-values <32 yielded test sensitivities of 95.2% (95%CI: 89.3–98.5%) in Utrecht and 98.0% (95%CI: 89.2–99.95%) in Aruba. Interpretation: In community-dwelling subjects with mild respiratory symptoms the Panbio™ COVID-19 Ag Rapid Test had 100% specificity, and a sensitivity above 95% for nasopharyngeal samples when using Ct-values <32 cycles as cut-off for RT-qPCR test positivity. Considering short turnaround times, user friendliness, low costs and opportunities for decentralized testing, this test can improve our efforts to control transmission of SARS-CoV-2

Integrated analysis of viral blips, residual viremia, and associated factors in people with HIV: Results from a retrospective cohort study

The etiology of viral blips is not yet fully elucidated. One of the hypotheses is that blips reflect variations in residual viremia (RV) near the detectability threshold. In this study, we evaluated whether RV is associated with viral blips and which factors are associated with RV. All treatment regimens in 2010–2020 consisting of two nucleos(-t)ide reverse transcriptase inhibitors and one anchor (integrase strand transfer inhibitor [INSTI], non-nucleoside reverse transcriptase inhibitor [NNRTI], or protease inhibitor [PI]) in people with HIV (PWH) were evaluated for RV (detectable viremia <50 cp/mL) and blips (isolated viral loads [VLs] 50–499 cp/mL between measurements <50 cp/mL). All medical records were reviewed and regimens in which a VL ≥ 50 cp/mL was deemed to result from non-adherence (based on the documented conclusion by the treating physician) were excluded. Factors associated with blips and RV were identified using generalized linear mixed models. In total, 24 518 VLs from 1658 PWH were analyzed. VLs were measured during INSTI- (n = 5119; 20.9%), PI- (n = 8935; 36.4%), and NNRTI-use (n = 10 464; 42.7%). VLs were categorized as blips in 1.4% (n = 332). The 24,186 non-blip VLs were RNAneg (no RV) (n = 15 326; 63.4%), 1–19 cp/mL (n = 6318; 26.1%), 20–49 cp/mL (n = 1620; 6.7%), or <50 cp/mL with an unknown RV level (n = 922; 3.8%). In 193/1658 PWH (11.6%), the RV level was RNAneg in all VLs assessed. RV 1–19 cp/mL and 20–49 cp/mL (vs. RNAneg) were significantly associated with subsequent viral blips (respective odds ratio 2.66 and 4.90 [95% confidence intervals: 1.98–3.58 and 3.41–7.04]). Zenith VL and use of PIs (vs. INSTIs/NNRTIs) were associated with higher RV and blip odds. This large cohort study showed that blips were associated with higher preceding RV. Both the anchor type and factors previously linked to the latent viral reservoir were associated with RV, suggesting blips having a multifactorial origin

Predictors of Treatment Adherence and Virological Failure Among People Living with HIV Receiving Antiretroviral Therapy in a South African Rural Community: A Sub-study of the ITREMA Randomised Clinical Trial

A large proportion of people living with HIV (PLHIV) in sub-Saharan Africa reside in rural areas. Knowledge of enablers and barriers of adherence to antiretroviral treatment (ART) in these populations is limited. We conducted a cohort study of 501 adult PLHIV on ART at a rural South African treatment facility as a sub-study of a clinical trial (ClinicalTrials.gov NCT03357588). Socio-economic, psychosocial and behavioral characteristics were assessed as covariates of self-reported adherence difficulties, suboptimal pill count adherence and virological failure during 96 weeks of follow-up. Male gender was an independent risk factor for all outcomes. Food insecurity was associated with virological failure in males. Depressive symptoms were independently associated with virological failure in both males and females. Household income and task-oriented coping score were protective against suboptimal pill-count adherence. These results underscore the impact of low household income, food insecurity and depression on outcomes of ART in rural settings and confirm other previously described risk factors. Recognition of these factors and targeted adherence support strategies may improve patient health and treatment outcomes

Clinical use of HIV integrase inhibitors : a systematic review and meta-analysis

Background: Optimal regimen choice of antiretroviral therapy is essential to achieve long-term clinical success. Integrase inhibitors have swiftly been adopted as part of current antiretroviral regimens. The purpose of this study was to review the evidence for integrase inhibitor use in clinical settings. Methods: MEDLINE and Web-of-Science were screened from April 2006 until November 2012, as were hand-searched scientific meeting proceedings. Multiple reviewers independently screened 1323 citations in duplicate to identify randomized controlled trials, nonrandomized controlled trials and cohort studies on integrase inhibitor use in clinical practice. Independent, duplicate data extraction and quality assessment were conducted. Results: 48 unique studies were included on the use of integrase inhibitors in antiretroviral therapy-naive patients and treatment-experienced patients with either virological failure or switching to integrase inhibitors while virologically suppressed. On the selected studies with comparable outcome measures and indication (n = 16), a meta-analysis was performed based on modified intention-to-treat (mITT), on-treatment (OT) and as-treated (AT) virological outcome data. In therapy-naive patients, favorable odds ratios (OR) for integrase inhibitor-based regimens were observed, (mITT OR 0.71, 95% CI 0.59-0.86). However, integrase inhibitors combined with protease inhibitors only did not result in a significant better virological outcome. Evidence further supported integrase inhibitor use following virological failure (mITT OR 0.27; 95% CI 0.11-0.66), but switching to integrase inhibitors from a high genetic barrier drug during successful treatment was not supported (mITT OR 1.43; 95% CI 0.89-2.31). Integrase inhibitor-based regimens result in similar immunological responses compared to other regimens. A low genetic barrier to drug-resistance development was observed for raltegravir and elvitegravir, but not for dolutegravir. Conclusion: In first-line therapy, integrase inhibitors are superior to other regimens. Integrase inhibitor use after virological failure is supported as well by the meta-analysis. Careful use is however warranted when replacing a high genetic barrier drug in treatment-experienced patients switching successful treatment

Virological failure after switch to long-acting cabotegravir and rilpivirine injectable therapy: an in-depth analysis

Background. Long-acting (LA) injectable therapy with cabotegravir (CAB) and rilpivirine (RPV) is currently used as maintenance treatment for human immunodeficiency virus type 1, and has a low risk for virological failure (VF). Although the risk is low, the circumstances and impact of VF in the real-world setting merit further evaluation. Methods. We performed an in-depth clinical, virological, and pharmacokinetic analysis on the reasons behind and the impact of VF during LA CAB/RPV therapy in 5 cases from the Netherlands. Genotypic resistance testing was performed after the occurrence of VF, and drug plasma (trough) concentrations were measured after VF was established and on any other samples to assess on-treatment drug levels. CAB and RPV drug levels that were below the first quartile of the population cutoff (≤Q1) were considered to be low. Results. Five cases who were eligible for LA CAB/RPV experienced VF despite a low predicted risk at baseline. Genotypic resistance testing revealed extensive selection of nonnucleoside reverse transcriptase inhibitor–associated mutations in all cases, and integrase strand transfer inhibitor mutations in 4 cases. All cases displayed low drug levels of either CAB, RPV, or both during the treatment course, likely contributing to the occurrence of VF. In 3 cases, we were able to identify the potential mechanisms behind these low drug levels. Conclusions. This is the first in-depth multiple case analysis of VF on LA CAB/RPV therapy in a real-world setting. Our observations stress the need to be aware for (evolving) risk factors and the yield of a comprehensive clinical, virological, and pharmacokinetic approach in case of failure

Trends and predictors of transmitted drug resistance (TDR) and clusters with TDR in a local Belgian HIV-1 epidemic

We aimed to study epidemic trends and predictors for transmitted drug resistance (TDR) in our region, its clinical impact and its association with transmission clusters. We included 778 patients from the AIDS Reference Center in Leuven (Belgium) diagnosed from 1998 to 2012. Resistance testing was performed using population-based sequencing and TDR was estimated using the WHO-2009 surveillance list. Phylogenetic analysis was performed using maximum likelihood and Bayesian techniques. The cohort was predominantly Belgian (58.4%), men who have sex with men (MSM) (42.8%), and chronically infected (86.5%). The overall TDR prevalence was 9.6% (95% confidence interval (CI): 7.7-11.9), 6.5% (CI: 5.0-8.5) for nucleoside reverse transcriptase inhibitors (NRTI), 2.2% (CI: 1.4-3.5) for non-NRTI (NNRTI), and 2.2% (CI: 1.4-3.5) for protease inhibitors. A significant parabolic trend of NNRTI-TDR was found (p = 0.019). Factors significantly associated with TDR in univariate analysis were male gender, Belgian origin, MSM, recent infection, transmission clusters and subtype B, while multivariate and Bayesian network analysis singled out subtype B as the most predictive factor of TDR. Subtype B was related with transmission clusters with TDR that included 42.6% of the TDR patients. Thanks to resistance testing, 83% of the patients with TDR who started therapy had undetectable viral load whereas half of the patients would likely have received a suboptimal therapy without this test. In conclusion, TDR remained stable and a NNRTI up-and-down trend was observed. While the presence of clusters with TDR is worrying, we could not identify an independent, non-sequence based predictor for TDR or transmission clusters with TDR that could help with guidelines or public health measures

Impaired SARS-CoV-2 specific T-cell response in patients with severe COVID-19

Cellular immune responses are of pivotal importance to understand SARS-CoV-2 pathogenicity. Using an enzyme-linked immunosorbent spot (ELISpot) interferon-γ release assay with wild-type spike, membrane and nucleocapsid peptide pools, we longitudinally characterized functional SARS-CoV-2 specific T-cell responses in a cohort of patients with mild, moderate and severe COVID-19. All patients were included before emergence of the Omicron (B.1.1.529) variant. Our most important finding was an impaired development of early IFN-γ-secreting virus-specific T-cells in severe patients compared to patients with moderate disease, indicating that absence of virus-specific cellular responses in the acute phase may act as a prognostic factor for severe disease. Remarkably, in addition to reactivity against the spike protein, a substantial proportion of the SARS-CoV-2 specific T-cell response was directed against the conserved membrane protein. This may be relevant for diagnostics and vaccine design, especially considering new variants with heavily mutated spike proteins. Our data further strengthen the hypothesis that dysregulated adaptive immunity plays a central role in COVID-19 immunopathogenesis

In-depth Characterization of Vaccine Breakthrough Infections With SARS-CoV-2 Among Health Care Workers in a Dutch Academic Medical Center

Severe acute respiratory syndrome coronavirus 2 infection after coronavirus disease 2019 vaccination raises concerns about the emergence of vaccine escape variants. Here we characterize 14 breakthrough infections among 5860 fully vaccinated Dutch health care workers ≥14 days after the final dose of vaccination with either BNT162b2, mRNA-1273, or Ad26.COV2.S. These breakthrough infections presented with regular B.1.1.7 (Alpha) and B.1.617.2 (Delta) variants and high viral loads, despite normal vaccine-induced B- and T-cell immune responses detected by live virus neutralization assays and ELISpot. High-risk exposure settings, such as in households, indicate a potential risk of viral transmission despite full vaccination

Impaired SARS-CoV-2 specific T-cell response in patients with severe COVID-19

Cellular immune responses are of pivotal importance to understand SARS-CoV-2 pathogenicity. Using an enzyme-linked immunosorbent spot (ELISpot) interferon-γ release assay with wild-type spike, membrane and nucleocapsid peptide pools, we longitudinally characterized functional SARS-CoV-2 specific T-cell responses in a cohort of patients with mild, moderate and severe COVID-19. All patients were included before emergence of the Omicron (B.1.1.529) variant. Our most important finding was an impaired development of early IFN-γ-secreting virus-specific T-cells in severe patients compared to patients with moderate disease, indicating that absence of virus-specific cellular responses in the acute phase may act as a prognostic factor for severe disease. Remarkably, in addition to reactivity against the spike protein, a substantial proportion of the SARS-CoV-2 specific T-cell response was directed against the conserved membrane protein. This may be relevant for diagnostics and vaccine design, especially considering new variants with heavily mutated spike proteins. Our data further strengthen the hypothesis that dysregulated adaptive immunity plays a central role in COVID-19 immunopathogenesis