There were three major data collections periods for the CHARTER study. During the initial study period, from 2004 - 2007 the CHARTER study’s aims were to determine how central and peripheral nervous system complications of HIV are affected by different histories and regimens of antiretroviral therapy.

To address these aims, the CHARTER study divided the study population into several substudies with common and differing assessments administered to participants within each substudy. Participants received comprehensive neuromedical, neurocognitive, and laboratory examinations, with a subset undergoing neuroimaging assessments. The specific study aims for this study period are outlined below:

CHARTER 2004 – 2007 Period Research aims:

1. To determine if patients taking HAART have reduced risk of CNS and PNS complications of HIV infection compared to those who are naïve to or have stopped prior ARV treatment. Epidemiologic studies suggest that HAART has reduced CNS complications of HIV, including opportunistic infections and cognitive impairment. This aim was designed to address this observation.
2. To determine if the CNS penetration profile of HAART is related to antiviral effects within the CNS (as measured by CSF HIV Ribonucleic Acid (RNA) levels) and to risk of HIV associated neurocognitive dysfunction (HAND) as measured by neurocognitive assessments. CNS complications of HIV appear to result from a combination of viral replication and secondary inflammatory damage. HIV replication in the CNS may be reflected better by its concentration in CSF than in plasma. Thus, viral suppression and prevention or reversal of CNS disease may depend on how well the ARV penetrates the blood-brain barrier (as reflected in CSF concentration) and how sensitive HIV in the CSF is to the drug regimen. Treatment of CNS HIV may require therapies directed at this compartment.
3. To determine if the mechanism of neurocognitive damage in late stage HIV disease differs from that in earlier stages of HIV disease. In later stages of HIV disease, the CNS becomes a compartment with "autonomous" HIV replication and evolution independent of the systemic (plasma/lymphoid) compartment. Evidence for this compartmentalization comes from a number of observations. Patients have discordant responses to ARVs in plasma and CSF. Reductions in CSF and plasma viral load are not parallel, with decline occurring more slowly in CSF than in plasma. This difference may reflect HIV originating from long-lived cells in the CNS (microglia and macrophages) in contrast to the short-lived lymphocytes that produce most of the plasma HIV. Phylogenetic analysis of the envelope, protease, and reverse transcriptase regions of multiple clones of HIV from plasma, lymph nodes, CSF, and brain show that the brain and CSF samples cluster separately from the systemic virions. Thus, these advanced patients, more so than those in earlier stages of HIV infection, may require penetration of ART for suppression of HIV in the CNS.
4. To measure the relationship of HIV replication within the CNS (as measured by CSF HIV RNA) and of immune competence (as measured by CD4 levels) to the prevalence and incidence of syndromic HIV associated neurocognitive dysfunction. Beneficial effects of ARVs in the CNS may result both from suppression of HIV replication and from immune reconstitution. In the pre-HAART era, higher CD4 counts correlated with lesser risk for neurocognitive complications. HAART-induced immune reconstitution as reflected by higher CD4 counts may reduce risk independent of its effect on HIV replication in the CNS.
5. To determine the prevalence of discordant ARV resistance in HIV plasma and CSF. Poor penetration with residual replication in the CNS has been postulated to promote evolution and selection of drug resistant HIV. Alternatively, because the resident viral population may be smaller and drug selection pressure may be less in the CNS, resistant strains may evolve there more slowly than in the plasma. A number of observations have shown that discordance in phenotypic or genotypic resistance can be demonstrated in HIV originating in the CNS and plasma. In addition, we have shown that ARV treatment may continue to suppress HIV in the CNS after loss of plasma suppression (see Virology Core).
6. To determine if the predictors and risk factors for peripheral neuropathy from HIV differ from that of d-drug (dideoxynucleoside HIV reverse transcriptase inhibitors; ddC, ddI, d4T) ARV neurotoxicity.

CHARTER 2004 – 2007 Supplementary Aims:

While the above aims were seen as central in guiding CHARTER, the original protocol also envisioned a number of additional questions which might be addressed through examinations of selected subsets of participants.

1. To determine how frequently psychotropic drugs prescribed to patients at different stages of disease and to elucidate the effects of such psychotropic medications on plasma concentrations of various ARVs.
2. To understand whether dideoxynucleoside analog reverse transcriptase inhibitors (e.g., Videx or didanosine (ddI) and Zerit or stavudine (d4T)) induce peripheral neuropathy by mitochondrial toxicity and to test whether serum lactic acidosis is a marker for disease.
3. To determine whether protease inhibitors (PI) increase risk of white matter disease in the brain.
4. To elucidate which patient characteristics place a person at risk for adverse neurocognitive effects of ART. For example, whether treatment with Cytochrome P450-3A, CYP2D6 utilizing ARV (e.g., Ritonavir (RTV)) results in enhanced behavioral toxicity in those receiving certain benzodiazepines and certain antidepressants.
5. To determine whether functional and physiological characteristics of peripheral neuropathy due to HIV and d-drugs can be prospectively identified using a novel clinical and quantitative battery.
6. To elucidate the risk factors for progression of peripheral neuropathy, with emphasis on transitions from neuropathy-free to neuropathy, and asymptomatic to symptomatic neuropathy and severe painful sensory neuropathies. Variables of interest include exposure to d-drug ARV, CD4 (current and nadir), HIV viral load (plasma and CSF), body weight, and anthropomorphic measurements.

The second phase of the CHARTER study began in 2007 and ended in 2010. This period focused on host genetics via SNP genotyping, viral genetics, peripheral neuropathy, and neuroimaging. Two exploratory aims were investigated during this period. Firstly, a subset of the original CHARTER participant population was identified for follow up in this period in the CHARTER Acute and Early Infection Substudy. These participants were well-characterized to understand the risk factors associated with the development of neurocognitive impairment. Secondly, the period from 2007 – 2010 sought to explore the relationship between new antiretroviral regimens and the development of HIV associated neurological complications. The specific aims of each element of this study are outlined below:

CHARTER 2007 – 2010 Period Research aims:

Host genetics research aims:

1. Validation of the genetic signature associated with HAND derived during the initial period of disease
2. To determine the association of genetic variability with longer term neurocognitive and peripheral neurological outcomes
3. To determine the pharmacogenetic associations for newer antiretroviral medications.

Viral genetics research aims:

1. To determine the prevalence of HIV env mutations in CSF and plasma among participants with and without HAND at enrollment
2. To determine the impact of viral genetics on the evolution of HAND in treated and untreated HIV infected participants over 7 years of follow-up
3. To provide an initial database of HIV env sequences that can be used as a reference for future studies aimed at defining the evolution of drug resistance in CSF virus to antiretroviral drugs that target viral binding and fusion

Peripheral neuropathy research aims:

1. To evaluate genetic susceptibility factors that may influence the expression of DSPN and d-drug neurotoxicity in patients with HIV infection
2. To determine the potential impact on peripheral neuropathy of long term exposure to HAART and its component antiretroviral agents
3. To detect potential increases in epidermal nerve fiber densities during a lengthy period of virological control in HAART-treated subjects
4. To evaluate whether lower epidermal nerve fiber densities predict the appearance of symptomatic neuropathy (SN) in those previously neuropathy-free, and worsening of SN in those with extant SN
5. To determine the potential neurotoxicity of protease inhibitor (PI)-containing HAART regimens
6. To better define the relationship between DSPN in HIV and the metabolic syndrome, a recognized complication of HAART and perhaps of HIV infection itself

Neuroimaging research aims:

1. To assess the longitudinal relationship between volume of white matter abnormality and markers of neuroinflammation
2. To investigate the relationship over time between volumes of abnormal white matter and white matter metabolites and executive functioning performance
3. To characterize the association between cortical damage and cumulative effects of neuroinflammation in the presence of high viral load
4. To investigate the performance impact of cortical tissue loss on learning and speed of information processing

Additional research aims:

1. To further our understanding of acute and early HIV infection, particularly the risk factors associated with the development of neurocognitive impairment
2. To determine the distribution of selected new antiretroviral in cerebrospinal fluid (CSF)
3. To determine the effectiveness of new antiretroviral and regimen types in reducing HIV ribonucleic acid (RNA) levels (viral load) in CSF
4. Specific aim 4: To determine the relationships between new antiretroviral medications and regimen types on the neurological and neurocognitive complications of HIV

The final phase of CHARTER data collection ran from September, 2010 – 2015. This period further refined the goals of the CHARTER study by focusing on extended follow-up of a select subset of participants. The goal of this period was to examine the effects of low-level viral replication in the CNS, predictors of neurocognitive and neuroimaging worsening, and the reversibility of nerve injury in the context of antiretroviral therapy. The specific aims and hypotheses are outlined below:

CHARTER 2010 – 2015 Period Research aims:

1. Specific aim 1: To explore the longitudinal neurological consequences of disproportionate HIV replication in the nervous system.
   Hypothesis: Participants with evidence of disproportionate HIV replication in the nervous system (defined as viral loads in CSF that are at least as high as those in plasma) will be more likely to have progressive neurological disease (as determined by neuropsychological testing and neuroimaging) than participants who do not have disproportionate HIV replication in the nervous system.
2. Specific aim 2: To examine cases that are receiving ART and undergoing cognitive decline or increasing white matter abnormalities based on neuroimaging in order to determine predictors of neurological injury.
   Hypothesis: In participants on ART, those having more evidence of white matter injury will have greater decline in neuropsychological functioning than those without any evidence of white matter injury. For our purposes, indicators of white matter injury include: increased volume of abnormal white matter signal and white matter volume loss on structural MRI; altered diffusion along fiber tracts, such as reduced fractional anisotropy, measured with diffusion tensor imaging (DTI); and changes in metabolite concentrations that may reflect injury in white matter, such as reduced N-acetyl aspartate (NAA) or increased choline (Cho), determined by MR spectroscopy

• In order to have a broad representation of participant characteristics, CHARTER was conducted at six performance sites nationally whose activities were coordinated by the University of California San Diego (UCSD), the principal grantee institution. The central data was curated by several technical, scientific, and administrative cores working cooperatively at UCSD. The six sites for clinical data collection were located at UCSD, University of Washington, Washington University in St. Louis, Icahn School of Medicine, Johns Hopkins University, and University of Texas Medical Branch.

• Subject recruitment began in September, 2003 at the six clinical data collection sites, and concluded in August, 2007. The study population was recruited with minimal exclusion criteria. Of the 2,016 potential participants who were screened for the study, 1,610 (79.9%) elected to participate in at least the cross-sectional portion of the study, with 723 recruiting from the cross-sectional sample into the longitudinal study. The subjects were recruited from the screening interview into the cross-sectional sample with few exclusion criteria. The main mechanism driving failure to recruit into the cross-sectional sample was self-selection out by screened participants who elected not to continue. In around 2% of cases, screened participants were not asked to continue into the cross-sectional sample because their screening interviewer doubted their ability to participate (e.g. when the screener felt they had the potential to be dangerous).

• The use of minimal exclusion criteria was in an effort to broadly represent the population of HIV-positive patients being followed in the site’s university based clinics, and thus care should be taken when generalizing the results outside of this setting. The CHARTER population may not be reflective of the HIV-positive population being served in other clinical settings or of the population who elect not to volunteer for research studies.

Participants were recruited into the CHARTER Cross-Sectional study from the screening interview. These participants completed a standardized set of Cross-Sectional study assessments at their baseline visit. This section outlines participant recruitment into the Cross-Sectional study and the Cross-Sectional neuromedical and neurobehavioral assessments performed.

Approximately 1,608 HIV-infected participants were enrolled into the CHARTER Cross-Sectional study during the period from 2003 – 2007. Participants were recruited from six participating university centers whose activities were coordinated by the University of California San Diego (UCSD), the principal grantee institution. The central data was curated by several technical, scientific, and administrative cores working cooperatively at UCSD. The number of participants recruited from each of the six participating clinical sites is summarized below:

• 
  Johns Hopkins University (Baltimore, MD) – 234 participants
• 
  Mt. Sinai School of Medicine (New York, NY) – 272 participants
• 
  University of California San Diego (San Diego, CA) – 290 participants
• 
  University of Texas Medical Branch (Galveston, TX) – 273 participants
• 
  University of Washington (Seattle, WA) – 267 participants
• 
  Washington University (St. Louis, MO) – 272 participants

Of the 2,016 potential participants who were screened for the study, 1,608 (~80%) elected to participate in at least the Cross-Sectional portion of the study. The participants recruited into CHARTER were at varying stages of disease and reported differing histories of antiretroviral treatment. CHARTER Cross-Sectional participants were recruited with relatively few exclusion criteria in an effort to broadly represent the population of HIV-positive patients being followed in the site’s university based clinics.

The subjects were recruited from the screening interview into the Cross-Sectional sample with relatively few exclusion criteria. The main mechanism driving failure to recruit into the cross-sectional sample was self-selection out by screened participants who elected not to continue. In around 2% of cases, screened participants were not asked to continue into the cross-sectional sample because their screening interviewer doubted their ability to participate (e.g. when the screener felt they had the potential to be dangerous).

The use of minimal exclusion criteria was in an effort to broadly represent the population of HIV-positive patients being followed in the site’s university based clinics, and thus care should be taken when generalizing results derived from CHARTER to populations outside the scope of this study. The CHARTER population may not be reflective of the HIV-positive population being served in other clinical settings or of the population who elect not to volunteer for research studies.

The likelihood of new neurocognitive complications, remission of such complications, or fluctuating patterns of complications were hypothesized to be related to several key variables. These variables include whether or not virus is suppressed in the CSF compartment, whether or not the individual is currently on treatment, the history of past treatment, and whether or not a particular neurological complication has already occurred. To determine the importance of these influences on changes in neurocognitive and neurological complications, CHARTER enrolled a subset of participants from the Cross-Sectional cohort in the Longitudinal ARV Substudy.

The CHARTER Longitudinal ARV substudy cohort consists of individuals characterized by the following variables:

1. History of HAART ARV regimen
2. Presence of detectable virus in the CSF
3. Presence or absence of neurocognitive complications at the beginning of the study

It was anticipated that careful six monthly observation of this cohort would reveal the degree to which HAART history and viral suppression predict near future onset of neurological complications; and secondly, would shed light on some of the intermediate steps in this link, for example, evolution of resistant virus in the CSF compartment.

Metabolic disorders such as lipoatrophy and centripetal fat re-distribution, elevated blood cholesterol and triglyceride levels (dyslipidemias), and insulin resistance (diabetes and impaired glucose tolerance) are commonly induced by HAART, but their impact on the CNS not been assessed systematically. Patients in the HAART era appear to have reduced rates of more severe cognitive deterioration. However, epidemiologic data showing the continuing incidence and prevalence of cognitive problems in HAART-treated patients raises concern about the possibility that HAART has protective effects through its antiviral effects, but is also toxic for the CNS in some patients. The primary aim was to relate lipoatrophy indicators to manifestations of HIV-associated neurocognitive dysfunction HAND and peripheral neuropathy.

Exposure to ARV regimens containing d-drugs ddC (zalcitabine, Hivid), ddI (didanosine, Videx EC) and d4T (stavudine, Zerit) is known to cause or contribute to peripheral neuropathy in some cases; however it is unclear how specific drugs, duration of exposure, and recency of exposure interact with individual characteristics as risk factors for developing distal sensory-predominant polyneuropathy (DSPN).

During the initial study period from 2004 – 2007, the CHARTER Longitudinal Peripheral Neuropathy Substudy was focused on understanding the short-term neurotoxic impact of ARV regimens containing d-drugs on distal sensory polyneuropathy. To this end, the Longitudinal Peripheral Neuropathy Substudy enrolled participants into three subgroups:

1. No clinical DSPN (no signs or symptoms of DSPN)
2. Asymptomatic DSPN (1 or more signs of DSPN, but no symptoms)
3. Symptomatic DSPN (both signs and symptoms present)

Severity of neuropathy was characterized according to the Total Neuropathy Score, including nerve conduction studies and quantitative sensory testing. Serum lactate levels were measured as a marker of mitochondrial dysfunction to investigate whether changes in lactate levels correlate with stabilization or improvement in neuropathy as measured by the Total Neuropathy Score.

During the study period from 2007 – 2015, the goals of the Longitudinal Peripheral Neuropathy Substudy were revised to emphasize the influence of long term antiretroviral drug exposure and genetic risk factors on the pathogenesis of neuropathy. To improve earlier ascertainment and longitudinal monitoring of nerve injury, intraepidermal nerve fiber (IENF) layer assessments (skin biopsies) were added to the CHARTER Longitudinal Peripheral Neuropathy substudy battery and performed from 2007 – 2010. The IENF technique, developed and validated over the past 5 to 10 years, provides a more direct measure of cutaneous innervation than clinical examinations or electrophysiological testing.

HIV penetrates the central nervous system shortly after infection, which may result in neurocognitive impairment in individuals who are otherwise in a physically “asymptomatic” phase of illness. The timing, characteristics, features and factors affecting the risk of neurocognitive impairment, and these factors’ relationship to antiretroviral therapy remain unclear. The goal of the Acute and Early Infection Substudy enrolled participants who were identified as either Acute or Early in their HIV infection, defined as those whose duration of infection was less than or equal to one year. These participants were enrolled into the Acute and Early Infection Substudy starting in 2007 and underwent the neuromedical and neurobehavioral protocols outlined below.

During the period from 2007 – 2010, the Viral Genetics Substudy was introduced to investigate viral genetics as predictors of neurocognitive dysfunction and the reversibility of neurocognitive dysfunction in the context of antiretroviral treatment. Over the course of the CHARTER study, new antiretroviral drugs that target the HIV entry process have been introduced. These medications are expected to elicit changes in the HIV env sequence as drug resistance develops. Data from several research groups also indicate that HIV env sequence features are correlated with neurocognitive impairment in vivo and virological phenotypes in vitro that may be relevant to infection in the central nervous system. The Viral Genetics Substudy also sought to address these issues and to establish a database of baseline env sequences for use in comparative studies of antiviral medications that target the HIV viral entry process.

Participants for the Viral Genetics Substudy were chosen based on the following criteria:

1. Plasma viral load > 400 copies/mL OR
2. Plasma and CSF viral load > 400 copies/mL

Approximately one-third of participants were co-enrolled in the CHARTER Longitudinal Peripheral Neuropathy Substudy and approximately one-third were co-enrolled in the CHARTER Neuroimaging Substudy.

Neuroimaging data were collected at the six clinical sites during the original CHARTER Contract Period (2004-2007), the CHARTER Option Period (2007-2010), and the CHARTER Extension Period (2010-2015).