The distribution of cerebral atherosclerosis is influenced by race and sex but not by other vascular risk factors. In our patient population, intracranial disease is as common a cause of cerebral ischemia as extracranial carotid disease.

Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants can greatly reduce mortality rates. However, current infant HIV-1 diagnostics cannot reliably be performed at the point of care, often delaying treatment and compromising its efficacy. Recombinase polymerase amplification (RPA) is a novel technology that is ideal for an HIV-1 diagnostic, as it amplifies target DNA in <20 min at a constant temperature, without the need for complex thermocycling equipment. Here we tested 63 HIV-1-specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome (long terminal repeat [LTR] and pol) and can reliably detect 3 copies of proviral DNA by the use of fluorescence detection and lateral-flow strip detection. These pol and LTR primers amplified 98.6% and 93%, respectively, of the diverse HIV-1 variants tested. This is the first example of an isothermal assay that consistently detects all of the major HIV-1 global subtypes.

Little is known about achievable levels of antiretroviral treatment (ART) adherence in resource-limited settings. We conducted a cross-sectional study of adherence among patients at Chris Hani Baragwanath Hospital's Adult HIV Clinic in Soweto, South Africa. Adherence was assessed using a 1-month, self-report questionnaire and was calculated as a ratio of doses taken to doses prescribed. The 66 patients studied had a mean age of 36.1 years, a median duration of ART use of 18 months, and an overall baseline median CD4(+) cell count of 200/mm(3) (IQR: 114-364). The adherence reported by these patients for the previous month was >95% for 58 patients (88%), 90-95% for 6 (9%) and, < 90% for 2 (3%). The main reasons given for missing doses were being away from home (30%), difficulty with the dosing schedules (23%), and running out of pills (12%). Adherence decreased considerably with fear of being stigmatized by the sexual partner (OR = 0.13 95%, CI 0.02-0.70). Plasma HIV RNA levels were <400 copies/ml in the majority of patients (73% of those with adherence >95% and 88% of patients with < or =95% adherence) and the overall median CD4(+) cell count rose to 324/mm(3) (IQR: 193-510). High adherence and viral suppression are achievable for a significant proportion of HIV-infected patients taking ART in a resource-limited area such as Soweto, South Africa. Strategies to maximize adherence in this setting should emphasize ready access to affordable and simple ART regimens, as well as HIV education programs to help increase awareness and decrease disease stigmatization.

Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25–43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS.

Cancer Consortium, and P30 AI027757 CFAR New During HIV infection, a reservoir of long-lived latently infected cells is established that persists during antiretroviral therapy (ART) and is the source of virus replication after treatment cessation. A better understanding of when viruses enter the HIV reservoir (reservoir seeding) will aid efforts to target these long-lived HIV infected cells during their establishment. We studied women infected at two different times with two genetically distinct HIV strains (called superinfection), and assessed the genetic relationship between sequences of the HIV strains that circulated throughout infection (pre-ART HIV RNA sequences) and the HIV strains that persisted in reservoir cells (HIV DNA sequences during ART). We estimated when HIV DNA sequences entered the reservoir by identifying the time the most genetically related HIV RNA sequence was detected. In most cases we observed that viruses in the reservoir included both the initial and superinfecting lineages, suggesting reservoir seeding occurs throughout HIV infection. However, the majority of HIV sequences entered the reservoir near the time of ART initiation, suggesting that novel strategies that aim to reduce reservoir size should focus on times immediately prior to ART.

Recombinase Polymerase Amplification (RPA) can be used to detect pathogen-specific DNA or RNA in under 20 minutes without the need for complex instrumentation. These properties enable its potential use in resource limited settings. However, there are concerns that deviations from the manufacturer’s protocol and/or storage conditions could influence its performance in low resource settings. RPA amplification relies upon viscous crowding agents for optimal nucleic acid amplification, and thus an interval mixing step after 3–6 minutes of incubation is recommended to distribute amplicons and improve performance. In this study we used a HIV-1 RPA assay to evaluate the effects of this mixing step on assay performance. A lack of mixing led to a longer time to amplification and inferior detection signal, compromising the sensitivity of the assay. However lowering the assay volume from 50 μL to 5 μL showed similar sensitivity with or without mixing. We present the first peer-reviewed study that assesses long term stability of RPA reagents without a cold chain. Reagents stored at −20°C, and 25°C for up to 12 weeks were able to detect 10 HIV-1 DNA copies. Reagents stored at 45°C for up to 3 weeks were able to detect 10 HIV-1 DNA copies, with reduced sensitivity only after >3 weeks at 45°C. Together our results show that reducing reaction volumes bypassed the need for the mixing step and that RPA reagents were stable even when stored for 3 weeks at very high temperatures.

A cross-sectional study of knowledge, attitudes, beliefs, and practices (KABPs) toward HIV and antiretroviral therapy (ART) was conducted in Soweto, South Africa, using a standardized validated questionnaire. Of 105 HIV clinic patients evaluated, 70% of whom were not on ART, 89% had good knowledge about the cause of HIV infection and 83% knew about modes of transmission. Fifty-nine percent reported they were not worried about ART side effects. Sixty-five percent agreed that missing ART doses can lead to disease progression. Ninety percent had disclosed their HIV serostatus to 1 or more persons, but only 62% of those with a current sexual partner reported having told that partner. Approximately 80% reported that if they were taking ART, they would not be worried about family or friends finding out. Forty-nine percent believed that ART can cure HIV, a belief that was associated with a low level of education (P<0.001). Overall, knowledge of the cause of HIV/AIDS, modes of transmission, and importance of ART adherence was good in our study population. Further research is warranted to assess the extent to which this knowledge and attendant attitudes predict ART adherence levels. The low rate of HIV serostatus disclosure to sexual partners calls for multidimensional interventions to reduce HIV-related stigma.

Drosophila imaginal discs are sac-like appendage primordia comprising apposed peripodial and columnar cell layers. Cell survival in disc columnar epithelia requires the secreted signal Decapentaplegic (DPP), which also acts as a gradient morphogen during pattern formation. The distribution mechanism by which secreted DPP mediates global cell survival and graded patterning is poorly understood. Here we report detection of DPP in the lumenal cavity between apposed peripodial and columnar cell layers of both wing and eye discs. We show that peripodial cell survival hinges upon DPP signal reception and implicate DPP-dependent viability of the peripodial epithelium in growth of the entire disc. These results are consistent with lumenal transmission of the DPP survival signal during imaginal disc development.