Antiretroviral choice and severe disease predict poorer neuropsychological outcomes in HIV+ children from Africa.

Background: The International Maternal Pediatric Adolescent AIDS Clinical Trials Network (IMPAACT) P1104s study evaluated neuropsychological outcomes over 96 weeks in children living with HIV (CLHIV) aged 5-11 years at 6 Sub-Saharan African sites to explore associations between HIV-illness related biomarkers and neuropsychological outcomes. Methods: Children living with HIV had participated in IMPAACT P1060, which compared efficacy of nevirapine versus lopinavir/ritonavir in children initiating ART at <3 years of age. At age 5-11, neuropsychological evaluations of KABC cognitive ability, TOVA attention-impulsivity and BOT-2 motor domains were assessed and repeated after 48 and 96 weeks. Clinical, antiretroviral therapy (ART) and laboratory (immunological and virological) parameters were used to predict neuropsychological outcomes using linear mixed-effects multivariable regression models, controlling for child and caregiver characteristics. Results: 246 CLHIV (45% male, mean age at initial neuropsychological evaluation 7.1 yrs [SD 1.2]) began ART at a median age 14.9 months (IQR 8.2, 25.2). Nadir CD4 percentage was 14.7% (IQR 11.0, 19.5); the median peak viral load (VL) was 750 000 copies/ml (IQR 366 000, 750 000) and 63% had ≥WHO stage 3 clinical disease; 164 (67%) were on lopinavir/ritonavir, 71 (29%) were on nevirapine and 7 (3%) were on efavirenz. Other antiretrovirals were similar. Nevirapine at P1104s study start or later was associated with poorer neuropsychological scores across all domains except Global Executive Composite, even when controlling for nadir CD4 percent and time-varying HIV VL. Other predictors of poorer scores in KABC domains included low birth weight, WHO stage 4 disease and serious illness history and elevated VL was associated with worse BOT-2 scores. Conclusion: Children receiving nevirapine had poorer neuropsychological scores than those on lopinavir/ritonavir. Antiretroviral choice might adversely impact neuropsychological performance. In addition, low birth weight and markers of severe HIV disease: advanced WHO clinical HIV disease, history of serious illness and an elevated VL, were associated with lower neuropsychological scores.

African Multi-Site 2-Year Neuropsychological Study of School-Age Children Perinatally Infected, Exposed, and Unexposed to Human Immunodeficiency Virus.

BACKGROUND: Children living with human immunodeficiency virus (HIV) are at neuropsychological risk for cognitive and motor dysfunction. However, few prospective, multi-site studies have evaluated neuropsychological outcomes longitudinally among perinatally infected African children who received early antiretroviral treatment (ART). METHODS: We enrolled 611 children aged 5 to 11 years at 6 sites (South Africa [3], Zimbabwe, Malawi, Uganda). Of these, there were 246 children living with HIV (HIV+) who were initiated on ART before 3 years of age in a prior clinical trial comparing nevirapine to lopinavir/ritonavir (International Maternal Pediatric Adolescent Acquired Immunodeficiency Syndrome Clinical Trials [IMPAACT] P1060); 183 age-matched, exposed but uninfected (HEU) children; and 182 unexposed and uninfected (HUU) children. They were compared across 3 assessment time points (Weeks 0, 48, and 96) on cognitive ability (Kaufman Assessment Battery for Children, second edition [KABC-II]), attention/impulsivity (Tests of Variables of Attention [TOVA]), motor proficiency (Bruininks-Oseretsky Test, second edition [BOT-2]), and on the Behavior Rating Inventory of Executive Function (BRIEF). The cohorts were compared using linear mixed models, adjusting for site, child's age and sex, and selected personal/family control variables. RESULTS: The HIV+ cohort performed significantly worse than the HEU and HUU cohorts for all KABC-II, TOVA, and BOT-2 performance outcomes across all 3 time points (P values < .001). The HUU and HEU cohorts were comparable. For the KABC-II planning/reasoning subtests, the HIV+ children showed less improvement over time than the HUU and HEU groups. The groups did not differ significantly on the BRIEF. CONCLUSIONS: Despite initiation of ART in early childhood and good viral suppression at the time of enrollment, the HIV+ group had poorer neuropsychological performance over time, with the gap progressively worsening in planning/reasoning. This can be debilitating for self-management in adolescence.

Health Professional Training and Capacity Strengthening Through International Academic Partnerships: The First Five Years of the Human Resources for Health Program in Rwanda.

BACKGROUND: The Rwanda Human Resources for Health Program (HRH Program) is a 7-year (2012-2019) health professional training initiative led by the Government of Rwanda with the goals of training a large, diverse, and competent health workforce and strengthening the capacity of academic institutions in Rwanda. METHODS: The data for this organizational case study was collected through official reports from the Rwanda Ministry of Health (MoH) and 22 participating US academic institutions, databases from the MoH and the College of Medicine and Health Sciences (CMHS) in Rwanda, and surveys completed by the co-authors. RESULTS: In the first 5 years of the HRH Program, a consortium of US academic institutions has deployed an average of 99 visiting faculty per year to support 22 training programs, which are on track to graduate almost 4600 students by 2019. The HRH Program has also built capacity within the CMHS by promoting the recruitment of Rwandan faculty and the establishment of additional partnerships and collaborations with the US academic institutions. CONCLUSION: The milestones achieved by the HRH Program have been substantial although some challenges persist. These challenges include adequately supporting the visiting faculty; pairing them with Rwandan faculty (twinning); ensuring strong communication and coordination among stakeholders; addressing mismatches in priorities between donors and implementers; the execution of a sustainability strategy; and the decision by one of the donors not to renew funding beyond March 2017. Over the next 2 academic years, it is critical for the sustainability of the 22 training programs supported by the HRH Program that the health-related Schools at the CMHS significantly scale up recruitment of new Rwandan faculty. The HRH Program can serve as a model for other training initiatives implemented in countries affected by a severe shortage of health professionals.

Neuropsychological performance in African children with HIV enrolled in a multisite antiretroviral clinical trial.

OBJECTIVE AND DESIGN: Children with HIV infection (HIV+) are at neuropsychological risk, but few studies have evaluated this at multiple sites in low-income and middle-income countries. We compared neuropsychological outcomes at enrollment (>5 years age) among HIV+, HIV perinatally exposed uninfected (HEU), and HIV unexposed uninfected (HUU) children from four sub-Saharan countries. METHODS: IMPAACT P1060 compared nevirapine versus lopinavir/ritonavir-based antiretroviral treatment (ART) in HIV-infected children 6-35 months of age. The present study (P1104s) enrolled P1060 children at 5-11 years of age and evaluated their neuropsychological performance over 2 years using the Kaufman Assessment Battery for Children, 2nd edition (KABC-II), Tests of Variables of Attention (TOVA), Bruininks-Oseretsky Test, 2nd edition (BOT-2), and parent-reported Behavior Rating Inventory of Executive Function (BRIEF). Cohorts were compared using generalized estimating equations least-squares means adjusted for site, child age and sex, and personal and social characteristics for child and caregiver. RESULTS: Six hundred and eleven (246 HIV+, 183 HEU, 182 HUU) of the 615 enrolled at six sites [South Africa (three), Zimbabwe, Malawi, Uganda] were available for analysis. Mean age was 7.2 years, 48% male, 69% in school. Unadjusted and adjusted comparisons were consistent. HIV+ children performed significantly worse than HEU and HUU cohorts on all KABC-II cognitive performance domains and on BOT-2 total motor proficiency (P < 0.001), but not on the BRIEF Global Executive Indices. HUU and HEU cohorts were comparable on cognitive outcomes. HIV+ children initiated on ART before 1 year of age had significantly better BRIEF evaluations (lower scores - fewer behavior problems), compared with those started after (P = 0.03). CONCLUSION: Significant cognitive deficits were documented among HIV+ children at school age, even when started on ART at an early age. Earlier HIV treatment, neuropsychological monitoring, and rehabilitative interventions are all needed. Subsequent testing for 2 more years will help further evaluate how HIV infection and exposure affect the developmental trajectory.

Drug-Based Lead Discovery: The Novel Ablative Antiretroviral Profile of Deferiprone in HIV-1-Infected Cells and in HIV-Infected Treatment-Naive Subjects of a Double-Blind, Placebo-Controlled, Randomized Exploratory Trial.

UNLABELLED: Antiretrovirals suppress HIV-1 production yet spare the sites of HIV-1 production, the HIV-1 DNA-harboring cells that evade immune detection and enable viral resistance on-drug and viral rebound off-drug. Therapeutic ablation of pathogenic cells markedly improves the outcome of many diseases. We extend this strategy to HIV-1 infection. Using drug-based lead discovery, we report the concentration threshold-dependent antiretroviral action of the medicinal chelator deferiprone and validate preclinical findings by a proof-of-concept double-blind trial. In isolate-infected primary cultures, supra-threshold concentrations during deferiprone monotherapy caused decline of HIV-1 RNA and HIV-1 DNA; did not allow viral breakthrough for up to 35 days on-drug, indicating resiliency against viral resistance; and prevented, for at least 87 days off-drug, viral rebound. Displaying a steep dose-effect curve, deferiprone produced infection-independent deficiency of hydroxylated hypusyl-eIF5A. However, unhydroxylated deoxyhypusyl-eIF5A accumulated particularly in HIV-infected cells; they preferentially underwent apoptotic DNA fragmentation. Since the threshold, ascertained at about 150 µM, is achievable in deferiprone-treated patients, we proceeded from cell culture directly to an exploratory trial. HIV-1 RNA was measured after 7 days on-drug and after 28 and 56 days off-drug. Subjects who attained supra-threshold concentrations in serum and completed the protocol of 17 oral doses, experienced a zidovudine-like decline of HIV-1 RNA on-drug that was maintained off-drug without statistically significant rebound for 8 weeks, over 670 times the drug's half-life and thus clearance from circulation. The uniform deferiprone threshold is in agreement with mapping of, and crystallographic 3D-data on, the active site of deoxyhypusyl hydroxylase (DOHH), the eIF5A-hydroxylating enzyme. We propose that deficiency of hypusine-containing eIF5A impedes the translation of mRNAs encoding proline cluster ('polyproline')-containing proteins, exemplified by Gag/p24, and facilitated by the excess of deoxyhypusine-containing eIF5A, releases the innate apoptotic defense of HIV-infected cells from viral blockade, thus depleting the cellular reservoir of HIV-1 DNA that drives breakthrough and rebound. TRIAL REGISTRATION: ClinicalTrial.gov NCT02191657.

Auditory Impairments in HIV-Infected Children.

OBJECTIVES: In a cross-sectional study of human immunodeficiency virus (HIV)-infected adults, the authors showed lower distortion product otoacoustic emissions (DPOAEs) in HIV+ individuals compared with controls as well as findings consistent with a central auditory processing deficit in HIV+ adults on antiretroviral therapy. The authors hypothesized that HIV+ children would also have a higher prevalence of abnormal central and peripheral hearing test results compared with HIV- controls. DESIGN: Pure-tone thresholds, DPOAEs, and tympanometry were performed on 244 subjects (131 HIV+ and 113 HIV- subjects). Thirty-five of the HIV+, and 3 of the HIV- subjects had a history of tuberculosis treatment. Gap detection results were available for 18 HIV- and 44 HIV+ children. Auditory brainstem response results were available for 72 HIV- and 72 HIV+ children. Data from ears with abnormal tympanograms were excluded. RESULTS: HIV+ subjects were significantly more likely to have abnormal tympanograms, histories of ear drainage, tuberculosis, or dizziness. All audiometric results were compared between groups using a two-way ANOVA with HIV status and ear drainage history as grouping variables. Mean audiometric thresholds, gap detection thresholds, and auditory brainstem response latencies did not differ between groups, although the HIV+ group had a higher proportion of individuals with a hearing loss >25 dB HL in the better ear. The HIV+ group had reduced DPOAE levels (p < 0.05) at multiple frequencies compared with HIV- subjects. No relationships were found between treatment regimens or delay in starting treatment and audiological parameters. CONCLUSIONS: As expected, children with HIV+ were more likely to have a history of ear drainage, and to have abnormal tympanograms. Similar to the adult findings, the HIV+ group did not show significantly reduced audiometric thresholds, but did have significantly lower DPOAE magnitudes. These data suggest that (1) HIV+ children often have middle ear damage which complicates understanding the direct effects of HIV on the hearing system, and (2) even when corrected for confounders DPOAEs were lower in the HIV+ group. Previous studies suggest ototoxicity from antiretroviral drugs is an unlikely cause of the reduced DPOAE magnitudes. Other possibilities include effects on efferent pathways connecting to outer hair cells or a direct effect of HIV on the cochlea.

Nutritional Deficiencies and Food Insecurity Among HIV-infected Children in Tanzania.

BACKGROUND: Poor nutrition has been associated with impaired immunity and accelerated disease progression in HIV-infected children. The aim of this study was to quantify the levels of nutrient intake in HIV-infected children and compare these to standard recommendations. METHODS: We surveyed HIV-infected Tanzanian children enrolled in a pediatric care program that provided routine nutritional counseling and vitamin supplementation. We obtained anthropometric measurements and determined 24-hour macronutrient and micronutrient intakes and food insecurity. Values were compared to recommended nutrient intakes based on age and gender. RESULTS: We interviewed 48 pairs of children and their caregiver(s). The age of the child ranged from 2-14 years; median age 6 and 60% female. The median weight-for-height z-score for children ≤ 5 years was 0.69 and BMI-for-age z-scores for children >5 was -0.84. Macronutrient evaluation showed that 29 (60%) children were deficient in dietary intake of energy; deficiency was more common in older children (p=0.004). Micronutrient evaluation shows that over half of study subjects were deficient in dietary intake of vitamin A, vitamin D, vitamin E, thiamine, riboflavin, niacin, folate, vitamin B12, and calcium. Food insecurity was reported by 20 (58%) caregivers. CONCLUSIONS AND PUBLIC HEALTH IMPLICATIONS: The diets of many HIV-infected children at a specialized treatment center in Tanzania do not meet recommended levels of macro-and micro-nutrients. Food insecurity was a contributory factor. Enhanced dietary counseling and provision of macro- and micro-nutrient supplements will be necessary to achieve optimal nutrition for most HIV-infected children in resource-poor regions.

Drug-induced reactivation of apoptosis abrogates HIV-1 infection.

HIV-1 blocks apoptosis, programmed cell death, an innate defense of cells against viral invasion. However, apoptosis can be selectively reactivated in HIV-infected cells by chemical agents that interfere with HIV-1 gene expression. We studied two globally used medicines, the topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Both medicines activated apoptosis preferentially in HIV-infected cells, suggesting that the drugs mediate escape from the viral suppression of defensive apoptosis. In infected H9 cells, ciclopirox and deferiprone enhanced mitochondrial membrane depolarization, initiating the intrinsic pathway of apoptosis to execution, as evidenced by caspase-3 activation, poly(ADP-ribose) polymerase proteolysis, DNA degradation, and apoptotic cell morphology. In isolate-infected peripheral blood mononuclear cells, ciclopirox collapsed HIV-1 production to the limit of viral protein and RNA detection. Despite prolonged monotherapy, ciclopirox did not elicit breakthrough. No viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir. Tests in mice predictive for cytotoxicity to human epithelia did not detect tissue damage or activation of apoptosis at a ciclopirox concentration that exceeded by orders of magnitude the concentration causing death of infected cells. We infer that ciclopirox and deferiprone act via therapeutic reclamation of apoptotic proficiency (TRAP) in HIV-infected cells and trigger their preferential elimination. Perturbations in viral protein expression suggest that the antiretroviral activity of both drugs stems from their ability to inhibit hydroxylation of cellular proteins essential for apoptosis and for viral infection, exemplified by eIF5A. Our findings identify ciclopirox and deferiprone as prototypes of selectively cytocidal antivirals that eliminate viral infection by destroying infected cells. A drug-based drug discovery program, based on these compounds, is warranted to determine the potential of such agents in clinical trials of HIV-infected patients.

Dynamics of the resting CD4(+) T-cell latent HIV reservoir in infants initiating HAART less than 6 months of age.

OBJECTIVES: Identification of HIV infection in exposed infants facilitates early therapy, which may limit viral reservoirs that maintain HIV infection under HAART. METHODS: The dynamics of the resting CD4 T-cell latent HIV reservoir was determined over the first 2 years of life in 17 HIV-infected infants initiating lopinavir/ritonavir-based HAART at a median age of 8.1 weeks and achieving adequate suppression of plasma viral load by 24 weeks. RESULTS: The resting CD4 T-cell latent HIV reservoir was detected in 12 of 14 (86%) infants tested at 24 weeks of HAART [median frequency 1.88 infectious units per million (IUPM); range <0.22 to 81.7), and remained measurable (median IUPM = 0.32; range <0.22 to 3.25) in six of 10 (60%) children retested at 96 weeks. The reservoir declined, from 24 to 96 weeks of HAART, at an estimated mean rate of 0.028 log10 IUPM/month, corresponding to a half-life of 11 months (95% confidence interval 6-30 months]. A strong relationship was found between the frequency of latently infected CD4 T cells at 96 weeks of HAART and time to first undetectable plasma viral load (Spearman r = 0.91, P < 0.001). CONCLUSION: Although the resting CD4 T-cell latent reservoir remains detectable over the first 2 years of HAART in a substantial proportion of infants, its size is associated with time to first undetectable viral load. To minimize HIV reservoirs in infants, rapid curtailment of viremia may limit HIV reservoirs and should be a therapeutic goal of early HAART in infants.

Long-term outcomes for HIV-infected infants less than 6 months of age at initiation of lopinavir/ritonavir combination antiretroviral therapy.

OBJECTIVE: To investigate the longitudinal pharmacokinetics, safety and efficacy of lopinavir/ritonavir (LPV/r) in HIV-infected infants initiating combination antiretroviral therapy (cART) between 2 weeks and 6 months of age. METHOD: A prospective, open-label, multicenter Phase I/II study of LPV/r-based cART at a dose of 300/75 mg/m(2)/dose LPV/r twice daily. Intensive pharmacokinetic sampling at 12 months of age and quarterly predose LPV concentrations were collected and safety, virologic and immunologic responses were monitored every 4-12 weeks up to 252 weeks. RESULTS: Thirty-one HIV-infected infants enrolled into two age cohorts, 14 days to <6 weeks and 6 weeks to <6 months; 29 completed ≥48 weeks of follow-up (median = 123 weeks, range 4-252). At 12 months of age, median LPV area under the curve was comparable for both age cohorts and similar to older children and adults. At week 48, 22 of 31 patients (71%) had HIV-1 RNA <400 copies/ml and 11 of 15 (73%) had <50 copies/ml; 29 of 31 achieved HIV-1 RNA <400 copies/ml on study treatment and 19 (66%) remained durably suppressed until the end of study; viral suppression correlated with a higher percentage of predose time points exceeding the LPV target of 1 µg/ml (92 vs. 71%, P = 0.002). CONCLUSION: LPV/r at 300/75 mg/m(2)/dose as part of a cART regimen resulted in viral suppression through 96 weeks of treatment in >65% of young infants. Due to initially low LPV exposure in infants <6 weeks of age, frequent dose adjustment for weight gain is advisable and consideration should be given to studying a higher dose for very young infants.

Assessing medication adherence of perinatally HIV-infected children using caregiver interviews.

Medication adherence is critical for children's HIV treatment success, but obtaining accurate assessments is challenging when complex measurement technologies are not feasible. Our goal was to evaluate a multidimensional adherence interview designed to improve on existing adherence measures. Data from caregivers (N = 126) of perinatally infected children were analyzed to determine the ability of the revised interview guide to detect potential treatment nonadherence. Questions related to viral load (VL) on a bivariate level included proportion of doses taken in the previous 3 days and 6 months, caregivers' knowledge of prescribed dosing frequencies, and caregivers' reports of problems associated with medication administration. VL was not associated with 3-day recall of missed doses. In multivariate analyses, only caregiver knowledge of prescribed dosing frequencies was uniquely associated with VL. Our modified interview appears to successfully identify family struggles with adherence and to have the capacity to help clinicians address medication adherence challenges.

Model for assessment of proficiency of human immunodeficiency virus type 1 sequencing-based genotypic antiretroviral assays.

Use of sequencing-based genotyping as a diagnostic assay for human immunodeficiency virus (HIV) antiretroviral resistance is increasing. Periodic evaluation of the proficiency of laboratories performing this assay should be established. It is important to identify components of the assay that influence the generation of reliable sequencing data and that should and can be monitored. A model was developed to determine what parameters were reasonable and feasible for assessing the performance of genotyping assays. Ten laboratories using the genotyping platform, HIV-1 Genotyping System (HGS) v. 1 and software versions 1.1 or 2.0, participated in two rounds of testing. For each round, each group was sent a panel consisting of three clinical samples to sequence in real time. Six months later, seven laboratories using the TRUGENE HIV-1 Genotyping Kit participated in a separate round, working with both panels at the same time. Analysis of the data showed that one main indicator of genotyping proficiency was achievement of > or =98% sequence homology of a sample tested to a group consensus sequence for that sample. A second was concordant identification of codons at sites identified with resistance mutations in the sample, although scoring of these criteria is still undetermined from this study. These criteria are applicable to all sequence-based genotyping platforms and have been used as a baseline for assessing the performance of genotyping for the determination of antiretroviral resistance in our ongoing proficiency program.

Evaluation of the editing process in human immunodeficiency virus type 1 genotyping.

Sequencing-based human immunodeficiency virus type 1 (HIV-1) genotyping assays require subjective interpretation (editing) of sequence data from multiple primers to form consensus sequences and identify antiretroviral drug resistance mutations. We assessed interlaboratory variations in editing and their impact on the recognition of resistance mutations. Six samples were analyzed in a central laboratory by using a research-use-only HIV-1 genotyping system previously produced by Applied Biosystems. The electronic files of individual primer sequences from the samples were sent to 10 laboratories to compare sequence editing strategies. Each sequence data set included sequences from seven primers spanning protease codons 1 to 99 and reverse transcriptase codons 1 to 320. Each laboratory generated a consensus sequence for each sample and completed a questionnaire about editing strategy. The amount of editing performed, the concordance of consensus sequences among the laboratories, and the identification of resistance mutations were evaluated. Sequence agreement was high among the laboratories despite wide variations in editing strategies. All laboratories identified 66 (88%) of 75 resistance mutations in the samples. Nonconcordant identifications were made for 9 (12%) of the 75 mutations, all of which required editing for identification. These results indicate a need for standardized editing guidelines in genotyping assays. Proficiency in editing should be assessed in training and included in quality control programs for HIV-1 genotyping.

Dendritic cell amplification of HIV type 1-specific CD8+ T cell responses in exposed, seronegative heterosexual women.

In the Heterosexual AIDS Transmission Study (HATS), the frequency of high-risk sexual activity and viral load in the seropositive partner were shown to correlate with HIV-1 transmission. However, these parameters could not account for the status of some exposed, seronegative (ESN) individuals who remained uninfected despite years of exposure. To test the hypothesis that antiviral immune responses are a correlate of nontransmission in this cohort, we developed two sensitive methods for assessing HIV-1-specific humoral and cell-mediated responses. To quantify T cell responses, autologous mature dendritic cells (DCs) were used as antigen-presenting cells to elicit HIV-1-specific IFN-gamma production by ELISPOT. Antibody responses to HIV-1 gp120 were assessed by combination immunoprecipitation-Western blot (IP-WB). Previous studies of this cohort, using limiting dilution analysis, did not reveal HIV-1-specific cytotoxic T lymphocyte activity. However, when autologous DCs were used to present HIV-1 antigens, T cells from three of eight ESN women (38%) responded by producing IFN-gamma. T cells from three of four seropositive partners responded to HIV-1 antigens, whereas five negative controls did not. The use of DCs as antigen-presenting cells increased sensitivity by 2- to 30-fold relative to standard ELISPOT. Using IP-WB, low levels of gp120-reactive antibodies were detected in plasma from 1 of 14 ESN women. These results support the hypothesis that HIV-1-specific T cell responses play a role in immune surveillance in this cohort of North American serodiscordant couples. This report also demonstrates the ability of dendritic cells to reveal T cell responses that might be overlooked by other methods.