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-derivednefclones 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 Iin 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'sR = 0.61,P = 0.0004) and replication-competent reservoir sizes (Spearman'sR = 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.IMPORTANCEWhile 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 apost hocanalysis 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.

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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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.

Background Parents/caregivers strongly influence their child’s experience in the pediatric emergency department (PED). Understanding their needs can improve families’ PED experience and care. Objectives To describe caregivers’ perceived emotional, practical, and informational needs in the PED and how well these needs were met Design/Methods This descriptive, cross-sectional survey with medical record review was conducted at 10 Canadian PEDs from October 2018 to March 2020, over a 1-week period every season, for a total of 4 weeks per site. Digital surveys available in English or French were completed by caregivers in the PED and 7 days after their visit. Health literacy was evaluated by the Newest Vital Sign tool. Results A total of 2005 caregivers participated; mean age was 37.8 (SD 7.7) years, and 74.3% (1462/1969) were mothers. The mean child age was 5.9 (5.1) years and 51.9 % (1040/2003) were male. Caregivers’ main spoken language was mostly English [72.6% (1425/1964)] or French [10.5% (206/1964)]. Over half of caregivers had a university/professional degree [51.0% (997/1956)]; 37.4% (746/1998) had possible/probable limited health literacy. 77.9% (1132/1454) of caregivers reported that their overall care needs were met. 71.7% (1081/1507) of caregivers reported that their emotional needs were met; they reported most emotional support by doctors [(35.6%, 535/1510)] and bedside nurses [(24.2%, 364/1507)]. The median (IQR) length of stay was 3.9 (2.6, 6.1) hours; 36.7% (546/1487) of caregivers felt they waited ‘too long’, and 77.8% (1158/1489) did not know how long they would have to wait. 11.7% of families were dissatisfied with the care updates provided. A safe PED was the top overall identified practical need [(45.1%, 602/1336)]. Internet access was the most important practical need throughout the visit [(28.1%, 378/1345)], while more space was the most reported [(49.8%, 696/1398)] need in the waiting room. The most desired improvements in the PED were better communication of wait times and delays [(41.1%, 745/1812)], shorter wait times [(38.8%, 699/1801)] and faster diagnosis [(24.9%, 449/1803)]. Conclusion Almost one quarter of Canadian caregivers report that their overall needs were not fully met when attending the PED. Safety during the PED visit was of utmost importance. Nationally, caregivers value shorter wait times, rapid diagnosis, and better communication about perceived delays. Top practical needs included internet access and more waiting room space. Creating innovative waiting systems, better methods to share wait time information, free internet access, and improved physical spaces could better address identified caregiver needs in the PED.