HIV Subtype and Nef-Mediated Immune Evasion Function Correlate with Viral Reservoir Size in Early-Treated Individuals.

The HIV accessory protein Nef modulates key immune evasion and pathogenic functions, and its encoding gene region exhibits high sequence diversity. Given the recent identification of early HIV-specific adaptive immune responses as novel correlates of HIV reservoir size, we hypothesized that viral factors that facilitate the evasion of such responses-namely, Nef genetic and functional diversity-might also influence reservoir establishment and/or persistence. We isolated baseline plasma HIV RNA-derived nef clones from 30 acute/early-infected individuals who participated in a clinical trial of early combination antiretroviral therapy (cART) (<6 months following infection) and assessed each Nef clone's ability to downregulate CD4 and human leukocyte antigen (HLA) class I in vitro We then explored the relationships between baseline clinical, immunological, and virological characteristics and the HIV reservoir size measured 48 weeks following initiation of suppressive cART (where the reservoir size was quantified in terms of the proviral DNA loads as well as the levels of replication-competent HIV in CD4+ T cells). Maximal within-host Nef-mediated downregulation of HLA, but not CD4, correlated positively with post-cART proviral DNA levels (Spearman's R = 0.61, P = 0.0004) and replication-competent reservoir sizes (Spearman's R = 0.36, P = 0.056) in univariable analyses. Furthermore, the Nef-mediated HLA downregulation function was retained in final multivariable models adjusting for established clinical and immunological correlates of reservoir size. Finally, HIV subtype B-infected persons (n = 25) harbored significantly larger viral reservoirs than non-subtype B-infected persons (2 infected with subtype CRF01_AE and 3 infected with subtype G). Our results highlight a potentially important role of viral factors-in particular, HIV subtype and accessory protein function-in modulating viral reservoir establishment and persistence.IMPORTANCE While combination antiretroviral therapies (cART) have transformed HIV infection into a chronic manageable condition, they do not act upon the latent HIV reservoir and are therefore not curative. As HIV cure or remission should be more readily achievable in individuals with smaller HIV reservoirs, achieving a deeper understanding of the clinical, immunological, and virological determinants of reservoir size is critical to eradication efforts. We performed a post hoc analysis of 30 participants of a clinical trial of early cART who had previously been assessed in detail for their clinical, immunological, and reservoir size characteristics. We observed that the HIV subtype and autologous Nef-mediated HLA downregulation function correlated with the viral reservoir size measured approximately 1 year post-cART initiation. Our findings highlight virological characteristics-both genetic and functional-as possible novel determinants of HIV reservoir establishment and persistence.

A national survey of children's experiences and needs when attending Canadian pediatric emergency departments.

BACKGROUND: Optimizing a child's emergency department (ED) experience positively impacts their memories and future healthcare interactions. Our objectives were to describe children's perspectives of their needs and experiences during their ED visit and relate this to their understanding of their condition. METHODS: 514 children, aged 7-17 years, and their caregivers presenting to 10 Canadian pediatric EDs completed a descriptive cross-sectional survey from 2018-2020. RESULTS: Median child age was 12.0 years (IQR 9.0-14.0); 56.5% (290/513) were female. 78.8% (398/505) reported adequate privacy during healthcare conversations and 78.3% (395/504) during examination. 69.5% (348/501) understood their diagnosis, 89.4% (355/397) the rationale for performed tests, and 67.2% (338/503) their treatment plan. Children felt well taken care of by nurses (90.9%, 457/503) and doctors (90.8%, 444/489). Overall, 94.8% (475/501) of children were happy with their ED visit. Predictors of a child better understanding their diagnosis included doctors talking directly to them (OR 2.21 [1.15, 4.28]), having someone answer questions and worries (OR 2.51 [1.26, 5.01]), and older age (OR 1.08 [1.01, 1.16]). Direct communication with a doctor (OR 2.08 [1.09, 3.99]) was associated with children better understanding their treatment, while greater fear/ 'being scared' at baseline (OR 0.59 [0.39, 0.89]) or at discharge (OR 0.46 [0.22, 0.96]) had the opposite effect. INTERPRETATION: While almost all children felt well taken care of and were happy with their visit, close to 1/3 did not understand their diagnosis or its management. Children's reported satisfaction in the ED should not be equated with understanding of their medical condition. Further, caution should be employed in using caregiver satisfaction as a proxy for children's satisfaction with their ED visit, as caregiver satisfaction is highly linked to having their own needs being met.

Family caregivers' emotional and communication needs in Canadian pediatric emergency departments.

OBJECTIVES: To describe the extent to which caregivers' emotional and communication needs were met during pediatric emergency department (PED) visits. Secondary objectives included describing the association of caregiver emotional needs, satisfaction with care, and comfort in caring for their child's illness at the time of discharge with demographic characteristics, caregiver experiences, and ED visit details. STUDY DESIGN: Electronic surveys with medical record review were deployed at ten Canadian PEDs from October 2018 -March 2020. A convenience sample of families with children <18 years presenting to a PED were enrolled, for one week every three months, for one year per site. Caregivers completed one in-PED survey and a follow-up survey, up to seven days post-visit. RESULTS: This study recruited 2005 caregivers who self-identified as mothers (74.3%, 1462/1969); mean age was 37.8 years (SD 7.7). 71.7% (1081/1507) of caregivers felt their emotional needs were met. 86.4% (1293/1496) identified communication with the doctor as good/very good and 83.4% (1249/1498) with their child's nurse. Caregiver involvement in their child's care was reported as good/very good 85.6% (1271/1485) of the time. 81.8% (1074/1313) of caregivers felt comfortable in caring for their child at home at the time of discharge. Lower caregiver anxiety scores, caregiver involvement in their child's care, satisfactory updates, and having questions adequately addressed positively impacted caregiver emotional needs and increased caregiver comfort in caring for their child's illness at home. CONCLUSION: Approximately 30% of caregivers presenting to PEDs have unmet emotional needs, over 15% had unmet communication needs, and 15% felt inadequately involved in their child's care. Family caregiver involvement in care and good communication from PED staff are key elements in improving overall patient experience and satisfaction.

Challenges with accessing health care for young children presumed to have malaria in the rural district of Butaleja, Uganda: a qualitative study.

OBJECTIVE: A qualitative study was conducted to gain insight into challenges reported by Butaleja households during a previous household survey. Specifically, this paper discusses heads of households' and caregivers' perceptions of challenges they face when seeking care for their very young children with fever presumed to be malaria. METHODS: Eleven focus groups (FGs) were carried out with household members (five with heads of households and six with household caregivers) residing in five sub-counties located across the district. Purposive sampling was used to ensure the sample represented the religious diversity and geographical distance from the peri-urban center of the district. Each FG consisted of five to six participants. The FGs were conducted at a community centre by two pairs of researchers residing in the district and who were fluent in both English and the local dialect of Lunyole. The discussions were recorded, translated, and transcribed. Transcripts were reviewed and coded with the assistance of QDA Miner (version 4.0) qualitative data management software, and analyzed using thematic content analysis. RESULTS: The FG discussions identified four major areas of challenges when managing acute febrile illness in their child under the age of five with presumed malaria (1) difficulties with getting to public health facilities due to long geographical distances and lack of affordable transportation; (2) poor service once at a public health facility, including denial of care, delay in treatment, and negative experiences with the staff; (3) difficulties with managing the child's illness at home, including challenges with keeping home-stock medicines and administering medicines as prescribed; and (4) constrained to use private outlets despite their shortcomings. CONCLUSIONS: Future interventions may need to look beyond the public health system to improve case management of childhood malaria at the community level in rural districts such as Butaleja. Given the difficulties with accessing quality private health outlets, there is a need to partner with the private sector to explore feasible models of community-based health insurance programs and expand the role of informal private providers.

Modulation of TCR-dependent NFAT signaling is impaired in HIV-1 Nef isolates from elite controllers.

HIV-1 Nef modulates the activation state of CD4+ T cells by altering signaling events elicited by the T cell receptor (TCR). Primary nef sequences exhibit extensive inter-individual diversity that influences their ability to downregulate CD4 and HLA class I; however, the impact of nef variation on modulation of T cell signaling is poorly characterized. Here, we measured TCR-mediated activation of NFAT transcription factor in the presence of nef alleles isolated from 45 elite controllers (EC) and 46 chronic progressors (CP). EC Nef clones displayed lower ability to inhibit NFAT signaling (median 87 [IQR 75-93]% relative to SF2 Nef) compared to CP clones (94 [IQR 89-98]%) (p < 0.001). Polymorphisms in Nef's N-terminal domain impaired its ability to inhibit NFAT signaling. Results indicate that primary nef alleles exhibit a range of abilities to modulate TCR-dependent NFAT signaling, implicating natural variation in this function as a potential contributor to differential HIV-1 pathogenesis.

In vitro functional assessment of natural HIV-1 group M Vpu sequences using a universal priming approach.

The HIV-1 accessory protein Vpu exhibits high inter- and intra- subtype genetic diversity that may influence Vpu function and possibly contribute to HIV-1 pathogenesis. However, scalable methods to evaluate genotype/phenotype relationships in natural Vpu sequences are limited, particularly those expressing the protein in CD4+ T-cells, the natural target of HIV-1 infection. A major impediment to assay scalability is the extensive genetic diversity within, and immediately upstream of, Vpu's initial 5' coding region, which has necessitated the design of oligonucleotide primers specific for each individual HIV-1 isolate (or subtype). To address this, we developed two universal forward primers, located in relatively conserved regions 38 and 90 bases upstream of Vpu, and a single universal reverse primer downstream of Vpu, which are predicted to cover the vast majority of global HIV-1 group M sequence diversity. We show that inclusion of up to 90 upstream bases of HIV-1 genomic sequence does not significantly influence in vitro Vpu expression or function when a Rev/Rev Response Element (RRE)-dependent expression system is used. We further assess the function of four diverse HIV-1 Vpu sequences, revealing reproducible and significant differences between them. Our approach represents a scalable option to measure the in vitro function of genetically diverse natural Vpu isolates in a CD4+ T-cell line.

Challenges with accessing health care for young children presumed to have malaria in the rural district of Butaleja, Uganda: a qualitative study

OBJECTIVE: A qualitative study was conducted to gain insight into challenges reported by Butaleja households during a previous household survey. Specifically, this paper discusses heads of households' and caregivers' perceptions of challenges they face when seeking care for their very young children with fever presumed to be malaria. METHODS: Eleven focus groups (FGs) were carried out with household members (five with heads of households and six with household caregivers) residing in five sub-counties located across the district. Purposive sampling was used to ensure the sample represented the religious diversity and geographical distance from the peri-urban center of the district. Each FG consisted of five to six participants. The FGs were conducted at a community centre by two pairs of researchers residing in the district and who were fluent in both English and the local dialect of Lunyole. The discussions were recorded, translated, and transcribed. Transcripts were reviewed and coded with the assistance of QDA Miner (version 4.0) qualitative data management software, and analyzed using thematic content analysis. RESULTS: The FG discussions identified four major areas of challenges when managing acute febrile illness in their child under the age of five with presumed malaria: (1) difficulties with getting to public health facilities due to long geographical distances and lack of affordable transportation; (2) poor service once at a public health facility, including denial of care, delay in treatment, and negative experiences with the staff; (3) difficulties with managing the child's illness at home, including challenges with keeping home-stock medicines and administering medicines as prescribed; and (4) constrained to use private outlets despite their shortcomings. CONCLUSIONS: Future interventions may need to look beyond the public health system to improve case management of childhood malaria at the community level in rural districts such as Butaleja. Given the difficulties with accessing quality private health outlets, there is a need to partner with the private sector to explore feasible models of community-based health insurance programs and expand the role of informal private providers

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