Management Principles in a Case of True-positive Loss of Neuromonitoring Signals during Scoliosis Surgery: A Case Report.

Introduction: Multimodal intraoperative neuromonitoring (IOM) is essential in scoliosis surgery. This is affected by misplaced instrumentation, cord trauma, hemodynamic instability, and anesthesia. We present an irreversible loss of IOM without identifiable intra-operative cause to highlight its occurrence and discuss post-operative investigations and management. Case Report: A 14-year-old girl with adolescent idiopathic scoliosis, no co-morbidities, and normal spinal magnetic resonance imaging (MRI) underwent posterior spinal fusion. During screw placement, bilateral motor evoked potentials (MEPs) and right somatosensory evoked potentials (SSEPs) were lost in the legs. All screws were removed with no evidence of cortical breach. Left leg responses gradually improved, but there was no recovery of right leg SSEPs or MEPs. Subsequently, the procedure was abandoned. The patient had reduced right leg strength (3/5) and sensation with the left leg was normal. Immediate post-operative spinal MRI identified no abnormality. Computed tomography (CT) showed no cortical breach with satisfactory pedicle screw tracts. Repeat MRI (day 7) showed high T2-signal within the cord at T11 indicating ischemia. Gradual neurological recovery occurred and on day 15, repeat neurophysiology found reproducible SSEPs and MagStim MEPs. The patient underwent revision posterior fusion with single rod correction without complication and IOM was maintained. By day 24, the patient had 5/5 power and normal sensation in both legs. Good scoliosis correction was achieved and maintained at 3-year follow-up. Conclusion: This patient represents a vascular event affecting the lower spinal cord and highlights the role of sequential imaging and pre-operative neurophysiology including MagStim in deciding when to proceed with revision surgery while reducing risk using conservative techniques.

Prediction of general practice licensure/certification examination performance by a measure of 'questionable' activities in clinical practice: a retrospective cohort study.

PURPOSE: 'Low-value' clinical care and medical services are 'questionable' activities, being more likely to cause harm than good or with disproportionately low benefit relative to cost. This study examined the predictive ability of the QUestionable In Training Clinical Activities Index (QUIT-CAI) for general practice (GP) registrars' (trainees') performance in Australian GP Fellowship examinations (licensure/certification examinations for independent GP). METHODS: The study was nested in ReCEnT, an ongoing cohort study in which Australian GP registrars document their in-consultation clinical practice. Outcome factors in analyses were individual registrars' scores on the three Fellowship examinations ('AKT', 'KFP', and 'OSCE' examinations) and pass/fail rates during 2012-21. Analyses used univariable and multivariable regression (linear or logistic, as appropriate). The study factor in each analysis was 'QUIT-CAI score percentage'-the percentage of times a registrar performed a QUIT-CAI clinical activity when 'at risk' (i.e. when managing a problem where performing a QUIT-CAI activity was a plausible option). RESULTS: A total of 1265, 1145, and 553 registrars sat Applied Knowledge Test, Key Features Problem, and Objective Structured Clinical Exam examinations, respectively. On multivariable analysis, higher QUIT-CAI score percentages (more questionable activities) were significantly associated with poorer Applied Knowledge Test scores (P = .001), poorer Key Features Problem scores (P = .003), and poorer Objective Structured Clinical Exam scores (P = .005). QUIT-CAI score percentages predicted Royal Australian College of General Practitioner exam failure [odds ratio 1.06 (95% CI 1.00, 1.12) per 1% increase in QUIT-CAI, P = .043]. CONCLUSION: Performing questionable clinical activities predicted poorer performance in the summative Fellowship examinations, thereby validating these examinations as measures of actual clinical performance (by our measure of clinical performance, which is relevant for a licensure/certification examination).

Video versus telephone for telehealth delivery: a cross-sectional study of Australian general practice trainees.

BACKGROUND: Remunerated telehealth consultations were introduced in Australia in 2020 in response to the COVID-19 pandemic. Videoconferencing has advantages over telephone-consulting, including improved diagnostic and decision-making accuracy. However, videoconferencing uptake in Australia has been low. This study aimed to establish prevalence and associations of video versus telephone consultations in Australian general practice (GP) registrars' practice. METHODS: A cross-sectional analysis of data from 2020 to 2021 (three 6-monthly data-collection rounds) from the Registrars Clinical Encounters in Training (ReCEnT) study. GP registrars record details of 60 consecutive consultations every 6-month term, for a total of 3 terms. Univariable and multivariable logistic regression were performed within the Generalized Estimating Equations framework with the outcome video versus telephone. RESULTS: 102,286 consultations were recorded by 1,168 registrars, with 21.4% of consultations performed via telehealth. Of these, telephone accounted for 96.6% (95% CI: 96.3-96.8%) and videoconferencing for 3.4% (95% CI: 3.2-3.7%). Statistically significant associations of using videoconferencing, compared to telephone, included longer consultation duration (OR 1.02, 95% CI: 1.01-1.03 per minute; and mean 14.9 versus 12.8 min), patients aged 0-14 years old (OR 1.29, 95% CI: 1.03-1.62, compared to age 15-34), patients new to the registrar (OR 1.19, 95% CI: 1.04-1.35), part-time registrars (OR 1.84, 95% CI: 1.08-3.15), and areas of less socioeconomic disadvantage (OR 1.27, 95% CI: 1.00-1.62 per decile). CONCLUSIONS: Registrars' telehealth consultations were mostly performed via telephone. Telephone use being associated with socioeconomic disadvantage has health equity implications. Future research should explore barriers to videoconferencing use and strategies to increase its uptake.

Exam prediction and the general Practice Registrar Competency Assessment Grid (GPR-CAG).

BACKGROUND: In GP training, identifying early predictors of poor summative examination performance can be challenging. We aimed to establish whether external clinical teaching visit (ECTV) performance, measured using a validated instrument (GP Registrar Competency Assessment Grid, GPR-CAG) is predictive of Royal Australian College of General Practitioners (RACGP) Fellowship examination performance. METHODS: A retrospective cohort study including GP registrars in New South Wales/Australian Capital Territory with ECTV data recorded during their first training term (GPT1), between 2014 and 2018, who attempted at least one Fellowship examination. Independent variables of interest included the four GPR-CAG factors assessed in GPT1 ('patient-centredness/caring', 'formulating hypotheses/management plans', 'professional responsibilities', 'physical examination skills'). Outcomes of interest included individual scores of the three summative examinations (Applied Knowledge Test (AKT); Key Feature Problem (KFP); and the Objective Structured Clinical Examination (OSCE)) and overall Pass/Fail status. Univariable and multivariable regression analyses were performed. RESULTS: Univariably, there were statistically significant associations (p < 0.01) between all four GPR-CAG factors and all four summative examination outcomes, except for 'formulating hypotheses/management plans' and OSCE score (p = 0.07). On multivariable analysis, each factor was significantly associated (p < 0.05) with at least one exam outcome, and 'physical examination skills' was significantly associated (p < 0.05) with all four exam outcomes. DISCUSSION: ECTV performance, via GPR-CAG scores, is predictive of RACGP Fellowship exam performance. The univariable findings highlight the pragmatic utility of ECTVs in flagging registrars who are at-risk of poor exam performance, facilitating early intervention. The multivariable associations of GPR-CAG scores and examination performance suggest that these scores provide predictive ability beyond that of other known predictors.

General practice registrars training part-time: a cross-sectional analysis of prevalence and associations.

While GPs are working fewer clinical hours and many GP trainees (registrars) do not foresee themselves working full-time in clinical practice, little is known of the epidemiology of registrars training part-time. We aimed to establish the prevalence of general practice part-time training (PTT), and part-time registrars' characteristics and practice patterns. A cross-sectional analysis was conducted of data from the Registrar Clinical Encounters in Training project, an ongoing cohort study of Australian GP registrars' clinical experiences over 60 consecutive consultations in each of three training terms. Univariable and multivariable logistic regression analyses were conducted with the outcome 'training part-time'. 1790 registrars contributed data for 4,135 registrar-terms and 241,945 clinical encounters. Nine hundred and twenty-two registrar-terms (22%, 95%CI:21%-24%) and 52,339 clinical encounters (22%, 95%CI:21%-22%) involved PTT. Factors associated with PTT were registrar characteristics - female gender, older age, in a later training stage, performing other regular medical work; practice characteristics - working in a higher socioeconomic status area; and patient characteristics - seeing more patients new to the registrar and seeing more patients from a non-English-speaking background. No consultation or consultation action factors were significantly associated with PTT. Registrars, practices, and patient associations have GP training implications. The lack of registrar consultation or consultation action associations suggests there may be limited impact of PTT on patient care.

'Low-value' clinical care in general practice: a cross-sectional analysis of low-value care in early-career GPs' practice.

Nonevidence-based and 'low-value' clinical care and medical services are 'questionable' clinical activities that are more likely to cause harm than good or whose benefit is disproportionately low compared with their cost. This study sought to establish general practitioner (GP), patient, practice, and in-consultation associations of an index of key nonevidence-based or low-value 'questionable' clinical practices. The study was nested in the Registrar Clinical Encounters in Training study-an ongoing (from 2010) cohort study in which Australian GP registrars (specialist GP trainees) record details of their in-consultation clinical and educational practice 6-monthly. The outcome factor in analyses, performed on Registrar Clinical Encounters in Training data from 2010 to 2020, was the score on the QUestionable In-Training Clinical Activities Index (QUIT-CAI), which incorporates recommendations of the Australian Choosing Wisely campaign. A cross-sectional analysis used negative binomial regression (with the model including an offset for the number of times the registrar was at risk of performing a questionable activity) to establish associations of QUIT-CAI scores. A total of 3206 individual registrars (response rate 89.9%) recorded 406 812 problems/diagnoses where they were at risk of performing a questionable activity. Of these problems/diagnoses, 15 560 (3.8%) involved questionable activities being performed. In multivariable analyses, higher QUIT-CAI scores (more questionable activities) were significantly associated with earlier registrar training terms: incidence rate ratios (IRRs) of 0.91 [95% confidence interval (CI) 0.87, 0.95] and 0.85 (95% CI 0.80, 0.90) for Term 2 and Term 3, respectively, compared to Term 1. Other significant associations of higher scores included the patient being new to the registrar (IRR 1.27; 95% CI 1.12, 1.45), the patient being of non-English-speaking background (IRR 1.24; 95% CI 1.04, 1.47), the practice being in a higher socioeconomic area decile (IRR 1.01; 95% CI 1.00, 1.02), small practice size (IRR 1.05; 95% CI 1.00, 1.10), shorter consultation duration (IRR 0.99 per minute; 95% CI 0.99, 1.00), and fewer problems addressed in the consultation (IRR 0.84; 95% CI 0.79, 0.89) for each additional problem]. Senior registrars' clinical practice entailed less 'questionable' clinical actions than junior registrars' practice. The association of lower QUIT-CAI scores with a measure of greater continuity of care (the patient not being new to the registrar) suggests that continuity should be supported and facilitated during GP training (and in established GPs' practice).

General practice trainees' telehealth use during the COVID-19 pandemic: a cross-sectional study.

BACKGROUND: Prompted by the COVID-19 pandemic, remuneration was introduced for Australian general practice telehealth consultations. General practitioner (GP) trainees' telehealth use is of clinical, educational, and policy importance. The aim of this study was to assess the prevalence and associations of telehealth versus face-to-face consultations amongst Australian GP registrars (vocational GP trainees). METHODS: Cross-sectional analysis of data from the Registrar Clinical Encounters in Training (ReCEnT) study, from 2020 to 2021 (three 6-month terms), including registrars in 3 of Australia's 9 Regional Training Organisations. In ReCEnT, GP registrars record details of 60 consecutive consultations, 6 monthly. The primary analysis used univariate and multivariable logistic regression, with outcome of whether the consultation was conducted via telehealth (phone and videoconference) or face-to-face. RESULTS: 1,168 registrars recorded details of 102,286 consultations, of which 21.4% (95% confidence interval [CI]: 21.1%-21.6%) were conducted via telehealth. Statistically significant associations of a telehealth consultation included shorter consultation duration (odds ratio [OR] 0.93, 95% CI: 0.93-0.94; and mean 12.9 versus 18.7 min); fewer problems addressed per consultation (OR 0.92, 95% CI: 0.87-0.97); being less likely to seek assistance from a supervisor (OR 0.86, 95% CI: 0.76-0.96) while being more likely to generate learning goals (OR 1.18, 95% CI: 1.02-1.37); and being more likely to arrange a follow-up consultation (OR 1.18, 95% CI: 1.02-1.35). CONCLUSIONS: That telehealth consultations were shorter, with higher rates of follow-up, has GP workforce/workload implications. That telehealth consultations were less likely to involve in-consultation supervisor support, but more likely to generate learning goals, has educational implications.

Unequal distribution of genetically-intact HIV-1 proviruses in cells expressing the immune checkpoint markers PD-1 and/or CTLA-4.

Introduction: HIV-1 persists in resting CD4+ T-cells despite antiretroviral therapy (ART). Determining the cell surface markers that enrich for genetically-intact HIV-1 genomes is vital in developing targeted curative strategies. Previous studies have found that HIV-1 proviral DNA is enriched in CD4+ T-cells expressing the immune checkpoint markers programmed cell death protein-1 (PD-1) or cytotoxic T-lymphocyte associated protein-4 (CTLA-4). There has also been some success in blocking these markers in an effort to reverse HIV-1 latency. However, it remains unclear whether cells expressing PD-1 and/or CTLA-4 are enriched for genetically-intact, and potentially replication-competent, HIV-1 genomes. Methods: We obtained peripheral blood from 16 HIV-1-infected participants, and paired lymph node from four of these participants, during effective ART. Memory CD4+ T-cells from either site were sorted into four populations: PD-1-CTLA-4- (double negative, DN), PD-1+CTLA-4- (PD-1+), PD-1-CTLA-4+ (CTLA-4+) and PD-1+CTLA-4+ (double positive, DP). We performed an exploratory study using the full-length individual proviral sequencing (FLIPS) assay to identify genetically-intact and defective genomes from each subset, as well as HIV-1 genomes with specific intact open reading frames (ORFs). Results and Discussion: In peripheral blood, we observed that proviruses found within PD-1+ cells are more likely to have intact ORFs for genes such as tat, rev and nef compared to DN, CTLA-4+ and DP cells, all of which may contribute to HIV-1 persistence. Conversely, we observed that CTLA-4 expression is a marker for cells harbouring HIV-1 provirus that is more likely to be defective, containing low levels of these intact ORFs. In the lymph node, we found evidence that CTLA-4+ cells contain lower levels of HIV-1 provirus compared to the other cell subsets. Importantly, however, we observed significant participant variation in the enrichment of HIV-1 proviruses with intact genomes or specific intact ORFs across these memory CD4+ T-cell subsets, and therefore consideration of additional cellular markers will likely be needed to consistently identify cells harbouring latent, and potentially replication-competent, HIV-1.

The Registrar Clinical Encounters in Training (ReCEnT) cohort study: updated protocol.

BACKGROUND: During vocational general practice training, the content of each trainee's (in Australia, registrars') in-consultation clinical experience is expected to entail a breadth of conditions that exemplify general practice, enabling registrars to gain competency in managing common clinical conditions and common clinical scenarios. Prior to the Registrar Clinical Encounters in Training (ReCEnT) project there was little research into the content of registrars' consultations despite its importance to quality of training. ReCEnT aims to document the consultation-based clinical and educational experiences of individual Australian registrars. METHODS: ReCEnT is an inception cohort study. It is comprised of closely interrelated research and educational components. Registrars are recruited by participating general practice regional training organisations. They provide demographic information about themselves, their skills, and their previous training. In each of three 6-month long general practice training terms they provide data about the practice where they work and collect data from 60 consecutive patient encounters using an online portal. Analysis of data uses standard techniques including linear and logistic regression modelling. The ReCEnT project has approval from the University of Newcastle Human Research Ethics Committee, Reference H-2009-0323. DISCUSSION: Strengths of the study are the granular detail of clinical practice relating to patient demographics, presenting problems/diagnoses, medication decisions, investigations requested, referrals made, procedures undertaken, follow-up arranged, learning goals generated, and in-consultation help sought; the linking of the above variables to the presenting problems/diagnoses to which they pertain; and a very high response rate. The study is limited by not having information regarding severity of illness, medical history of the patient, full medication regimens, or patient compliance to clinical decisions made at the consultation. Data is analysed using standard techniques to answer research questions that can be categorised as: mapping analyses of clinical exposure; exploratory analyses of associations of clinical exposure; mapping and exploratory analyses of educational actions; mapping and exploratory analyses of other outcomes; longitudinal 'within-registrar' analyses; longitudinal 'within-program' analyses; testing efficacy of educational interventions; and analyses of ReCEnT data together with data from other sources. The study enables identification of training needs and translation of subsequent evidence-based educational innovations into specialist training of general practitioners.

Markers of Immune Activation and Inflammation Are Associated with Higher Levels of Genetically-Intact HIV in HIV-HBV Co-Infected Individuals.

Co-infection with hepatitis B (HBV) and human immunodeficiency virus (HIV) increases overall and liver-related mortality. In order to identify interactions between these two viruses in vivo, full-length HIV proviruses were sequenced from a cohort of HIV-HBV co-infected participants and from a cohort of HIV mono-infected participants recruited from Bangkok, Thailand, both before the initiation of antiretroviral therapy (ART) and after at least 2 years of ART. The co-infected individuals were found to have higher levels of genetically-intact HIV proviruses than did mono-infected individuals pre-therapy. In these co-infected individuals, higher levels of genetically-intact HIV proviruses or proviral genetic-diversity were also associated with higher levels of sCD14 and CXCL10, suggesting that immune activation is linked to more genetically-intact HIV proviruses. Three years of ART decreased the overall level of HIV proviruses, with fewer genetically-intact proviruses being identified in co-infected versus mono-infected individuals. However, ART increased the frequency of certain genetic defects within proviruses and the expansion of identical HIV sequences. IMPORTANCE With the increased availability and efficacy of ART, co-morbidities are now one of the leading causes of death in HIV-positive individuals. One of these co-morbidities is co-infection with HBV. However, co-infections are still relatively understudied, especially in countries where such co-infections are endemic. Furthermore, these countries have different subtypes of HIV circulating than the commonly studied HIV subtype B. We believe that our study serves this understudied niche and provides a novel approach to investigating the impact of HBV co-infection on HIV infection. We examine co-infection at the molecular level in order to investigate indirect associations between the two viruses through their interactions with the immune system. We demonstrate that increased immune inflammation and activation in HBV co-infected individuals is associated with higher HIV viremia and an increased number of genetically-intact HIV proviruses in peripheral blood cells. This leads us to hypothesize that inflammation could be a driver in the increased mortality rate of HIV-HBV co-infected individuals.

Telehealth for Australian general practice: The present and the future.

BACKGROUND: In March 2020, 56 temporary Medicare Benefits Schedule telehealth item numbers were introduced for Australian general practitioners (GPs) in response to COVID-19. Telehealth is now a permanent part of Australian primary care and, as such, an adequate understanding of the benefits, barriers and facilitators is essential for GPs. OBJECTIVE: The aim of this article is to examine the use of telehealth in general practice in Australia and to explore the benefits, barriers and facilitators to performing telehealth consultations. A narrative review was performed. DISCUSSION: Benefits of telehealth include increased access to healthcare and reduced risk of COVID-19 transmission. Barriers can include lack of technological infrastructure, limitations to performing physical examination and concerns regarding privacy and confidentiality. Facilitators include tailored GP training and sustainable funding models. Further research and training are needed to ensure that telehealth is used optimally and equitably in Australia.

Antiretroviral Initiation at ≥800 CD4+ Cells/mm3 Associated With Lower Human Immunodeficiency Virus Reservoir Size.

BACKGROUND: Identifying factors that determine the frequency of latently infected CD4+ T cells on antiretroviral therapy (ART) may inform strategies for human immunodeficiency virus (HIV) cure. We investigated the role of CD4+ count at ART initiation for HIV persistence on ART. METHODS: Among participants of the Strategic Timing of Antiretroviral Treatment Study, we enrolled people with HIV (PWH) who initiated ART with CD4+ T-cell counts of 500-599, 600-799, or ≥ 800 cells/mm3. After 36-44 months on ART, the levels of total HIV-DNA, cell-associated unspliced HIV-RNA (CA-US HIV-RNA), and two-long terminal repeat HIV-DNA in CD4+ T cells were quantified and plasma HIV-RNA was measured by single-copy assay. We measured T-cell expression of Human Leucocyte Antigen-DR Isotype (HLA-DR), programmed death-1, and phosphorylated signal transducer and activator of transcription-5 (pSTAT5). Virological and immunological measures were compared across CD4+ strata. RESULTS: We enrolled 146 PWH, 36 in the 500-599, 60 in the 600-799, and 50 in the ≥ 800 CD4 strata. After 36-44 months of ART, total HIV-DNA, plasma HIV-RNA, and HLA-DR expression were significantly lower in PWH with CD4+ T-cell count ≥ 800 cells/mm3 at ART initiation compared with 600-799 or 500-599 cells/mm3. The median level of HIV-DNA after 36-44 months of ART was lower by 75% in participants initiating ART with ≥ 800 vs 500-599 cells/mm3 (median [interquartile range]: 16.3 [7.0-117.6] vs 68.4 [13.7-213.1] copies/million cells, respectively). Higher pSTAT5 expression significantly correlated with lower levels of HIV-DNA and CA-US HIV-RNA. Virological measures were significantly lower in females. CONCLUSIONS: Initiating ART with a CD4+ count ≥ 800 cells/mm3 compared with 600-799 or 500-599 cells/mm3 was associated with achieving a substantially smaller HIV reservoir on ART.

Plasma-Derived HIV-1 Virions Contain Considerable Levels of Defective Genomes.

Genetically-characterizing full-length HIV-1 RNA is critical for identifying genetically-intact genomes and for comparing these RNA genomes to proviral DNA. We have developed a method for sequencing plasma-derived RNA using long-range sequencing (PRLS assay; ∼8.3 kb from gag to the 3' end or ∼5 kb from integrase to the 3' end). We employed the gag-3' PRLS assay to sequence HIV-1 RNA genomes from ART-naive participants during acute/early infection (n = 6) or chronic infection (n = 2). On average, only 65% of plasma-derived genomes were genetically-intact. Defects were found in all genomic regions but were concentrated in env and pol. We compared these genomes to near-full-length proviral sequences from paired peripheral blood mononuclear cell (PBMC) samples for the acute/early group and found that near-identical (>99.98% identical) sequences were identified only during acute infection. For three participants who initiated therapy during acute infection, we used the int-3' PRLS assay to sequence plasma-derived genomes from an analytical treatment interruption and identified 100% identical genomes between pretherapy and rebound time points. The PRLS assay provides a new level of sensitivity for understanding the genetic composition of plasma-derived HIV-1 RNA from viremic individuals either pretherapy or after treatment interruption, which will be invaluable in assessing possible HIV-1 curative strategies. IMPORTANCE We developed novel plasma-derived RNA using long-range sequencing assays (PRLS assay; 8.3 kb, gag-3', and 5.0 kb, int-3'). Employing the gag-3' PRLS assay, we found that 26% to 51% of plasma-derived genomes are genetically-defective, largely as a result of frameshift mutations and deletions. These genetic defects were concentrated in the env region compared to gag and pol, likely a reflection of viral immune escape in env during untreated HIV-1 infection. Employing the int-3' PRLS assay, we found that analytical treatment interruption (ATI) plasma-derived sequences were identical and genetically-intact. Several sequences from the ATI plasma samples were identical to viral sequences from pretherapy plasma and PBMC samples, indicating that HIV-1 reservoirs established prior to therapy contribute to viral rebound during an ATI. Therefore, near-full-length sequencing of HIV-1 particles is required to gain an accurate picture of the genetic landscape of plasma HIV-1 virions in studies of HIV-1 replication and persistence.

The HIV-1 proviral landscape reveals that Nef contributes to HIV-1 persistence in effector memory CD4+ T cells.

Despite long-term antiretroviral therapy (ART), HIV-1 persists within a reservoir of CD4+ T cells that contribute to viral rebound if treatment is interrupted. Identifying the cellular populations that contribute to the HIV-1 reservoir and understanding the mechanisms of viral persistence are necessary to achieve an effective cure. In this regard, through Full-Length Individual Proviral Sequencing, we observed that the HIV-1 proviral landscape was different and changed with time on ART across naive and memory CD4+ T cell subsets isolated from 24 participants. We found that the proportion of genetically intact HIV-1 proviruses was higher and persisted over time in effector memory CD4+ T cells when compared with naive, central, and transitional memory CD4+ T cells. Interestingly, we found that escape mutations remained stable over time within effector memory T cells during therapy. Finally, we provided evidence that Nef plays a role in the persistence of genetically intact HIV-1. These findings posit effector memory T cells as a key component of the HIV-1 reservoir and suggest Nef as an attractive therapeutic target.

Cellular Activation, Differentiation, and Proliferation Influence the Dynamics of Genetically Intact Proviruses Over Time.

Human immunodeficiency virus (HIV) persists in cells despite antiretroviral therapy; however, the influence of cellular mechanisms such as activation, differentiation, and proliferation upon the distribution of proviruses over time is unclear. To address this, we used full-length sequencing to examine proviruses within memory CD4+ T-cell subsets longitudinally in 8 participants. Over time, the odds of identifying a provirus increased in effector and decreased in transitional memory cells. In all subsets, more activated (HLA-DR-expressing) cells contained a higher frequency of intact provirus, as did more differentiated cells such as transitional and effector memory subsets. The proportion of genetically identical proviruses increased over time, indicating that cellular proliferation was maintaining the persistent reservoir; however, the number of genetically identical proviral clusters in each subset was stable. As such, key biological processes of activation, differentiation, and proliferation influence the dynamics of the HIV reservoir and must be considered during the development of any immune intervention.

Neurotoxicity with high-dose disulfiram and vorinostat used for HIV latency reversal.

OBJECTIVE: The aim of this study was to examine whether administering both vorinostat and disulfiram to people with HIV (PWH) on antiretroviral therapy (ART) is well tolerated and can enhance HIV latency reversal. DESIGN: Vorinostat and disulfiram can increase HIV transcription in PWH on ART. Together, these agents may lead to significant HIV latency reversal. METHODS: Virologically suppressed PWH on ART received disulfiram 2000 mg daily for 28 days and vorinostat 400 mg daily on days 8-10 and 22-24. The primary endpoint was plasma HIV RNA on day 11 relative to baseline using a single copy assay. Assessments included cell-associated unspliced RNA as a marker of latency reversal, HIV DNA in CD4+ T-cells, plasma HIV RNA, and plasma concentrations of ART, vorinostat, and disulfiram. RESULTS: The first two participants (P1 and P2) experienced grade 3 neurotoxicity leading to trial suspension. After 24 days, P1 presented with confusion, lethargy, and ataxia having stopped disulfiram and ART. Symptoms resolved by day 29. After 11 days, P2 presented with paranoia, emotional lability, lethargy, ataxia, and study drugs were ceased. Symptoms resolved by day 23. CA-US RNA increased by 1.4-fold and 1.3-fold for P1 and P2 respectively. Plasma HIV RNA was detectable from day 8 to 37 (peak 81 copies ml-1) for P2 but was not increased in P1 Antiretroviral levels were therapeutic and neuronal injury markers were elevated in P1. CONCLUSION: The combination of prolonged high-dose disulfiram and vorinostat was not safe in PWH on ART and should not be pursued despite evidence of latency reversal.

HIV-1 Genomes Are Enriched in Memory CD4+ T-Cells with Short Half-Lives.

Future HIV-1 curative therapies require a thorough understanding of the distribution of genetically-intact HIV-1 within T-cell subsets during antiretroviral therapy (ART) and the cellular mechanisms that maintain this reservoir. Therefore, we sequenced near-full-length HIV-1 genomes and identified genetically-intact and genetically-defective genomes from resting naive, stem-cell memory, central memory, transitional memory, effector memory, and terminally-differentiated CD4+ T-cells with known cellular half-lives from 11 participants on ART. We find that a higher infection frequency with any HIV-1 genome was significantly associated with a shorter cellular half-life, such as transitional and effector memory cells. A similar enrichment of genetically-intact provirus was observed in these cells with relatively shorter half-lives. We found that effector memory and terminally-differentiated cells also had significantly higher levels of expansions of genetically-identical sequences, while only transitional and effector memory cells contained genetically-intact proviruses that were part of a cluster of identical sequences. Expansions of identical sequences were used to infer cellular proliferation from clonal expansion. Altogether, this indicates that specific cellular mechanisms such as short half-life and proliferative potential contribute to the persistence of genetically-intact HIV-1. IMPORTANCE The design of future HIV-1 curative therapies requires a more thorough understanding of the distribution of genetically-intact HIV-1 within T-cell subsets as well as the cellular mechanisms that maintain this reservoir. These genetically-intact and presumably replication-competent proviruses make up the latent HIV-1 reservoir. Our investigations into the possible cellular mechanisms maintaining the HIV-1 reservoir in different T-cell subsets have revealed a link between the half-lives of T-cells and the level of proviruses they contain. Taken together, we believe our study shows that more differentiated and proliferative cells, such as transitional and effector memory T-cells, contain the highest levels of genetically-intact proviruses, and the rapid turnover rate of these cells contributes to the expansion of genetically-intact proviruses within them. Therefore, our study delivers an in-depth assessment of the cellular mechanisms, such as cellular proliferation and half-life, that contribute to and maintain the latent HIV-1 reservoir.