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Unlabelled: HIV infection treatment strategies have historically defined effectiveness through measuring patient plasma HIV RNA. While combined antiretroviral therapy (cART) can reduce plasma viral load (pVL) to undetectable levels, the degree that HIV is eliminated from other anatomical sites remains unclear. We investigated the HIV DNA levels in 229 varied autopsy tissues from 20 HIV-positive (HIV(+)) cART-treated study participants with low or undetectable plasma VL and cerebrospinal fluid (CSF) VL prior to death who were enrolled in the National Neurological AIDS Bank (NNAB) longitudinal study and autopsy cohort. Extensive medical histories were obtained for each participant. Autopsy specimens, including at least six brain and nonbrain tissues per participant, were reviewed by study pathologists. HIV DNA, measured in tissues by quantitative and droplet digital PCR, was identified in 48/87 brain tissues and 82/142 nonbrain tissues at levels >200 HIV copies/million cell equivalents. No participant was found to be completely free of tissue HIV. Parallel sequencing studies from some tissues recovered intact HIV DNA and RNA. Abnormal histological findings were identified in all participants, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. All brain tissues demonstrated some degree of pathology. Ninety-five percent of participants had some degree of atherosclerosis, and 75% of participants died with cancer. This study assists in characterizing the anatomical locations of HIV, in particular, macrophage-rich tissues, such as the central nervous system (CNS) and testis. Additional studies are needed to determine if the HIV recovered from tissues promotes the pathogenesis of inflammatory diseases, such as HIV-associated neurocognitive disorders, cancer, and atherosclerosis., Importance: It is well-known that combined antiretroviral therapy (cART) can reduce plasma HIV to undetectable levels; however, cART cannot completely clear HIV infection. An ongoing question is, "Where is HIV hiding?" A well-studied HIV reservoir is "resting" T cells, which can be isolated from blood products and succumb to cART once activated. Less-studied reservoirs are anatomical tissue samples, which have unknown cART penetration, contain a comparably diverse spectrum of potentially HIV-infected immune cells, and are important since <2% of body lymphocytes actually reside in blood. We examined 229 varied autopsy specimens from 20 HIV(+) participants who died while on cART and identified that >50% of tissues were HIV infected. Additionally, we identified considerable pathology in participants' tissues, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. This study substantiates that tissue-associated HIV is present despite cART and can inform future studies into HIV persistence., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Inequities in the incidence of HIV infection and AIDS with continued persistence of HIV-associated neurocognitive disorders (HAND) exist in populations in Hawaii (HI) and Puerto Rico (PR). We previously reported that peripheral monocyte HIV DNA levels are high in patients in Hawaii with HAND and we now hypothesize that similar findings would be observed in the cerebrospinal fluid (CSF) cellular subsets. Cerebrospinal fluid cells were obtained from patients from PR and HI undergoing neurocognitive testing and sorted into monocytes (CD14+) and lymphocytes (CD14-) and HIV DNA was measured. From six PR subjects (three HAND, three normal cognition, NC) and six HI subjects (three HAND, three NC), HIV DNA burden in CD14+ cells was higher in HAND than NC patients; NC patients had higher HIV DNA burden in CD14-cells versus HAND. Differences in HIV DNA burden in particular CSF cellular subsets suggest that HIV DNA burden may play a role in HAND neuropathogenesis.

Objectives: Cognitive impairment remains frequent in HIV, despite combination antiretroviral therapy (cART). Leading theories implicate peripheral monocyte HIV DNA reservoirs as a mechanism for spread of the virus to the brain. These reservoirs remain present despite cART. The objective of this study was to determine if the level of HIV DNA in CD14(+) enriched monocytes predicted cognitive impairment and brain injury., Methods: We enrolled 61 cART-naïve HIV-infected Thais in a prospective study and measured HIV DNA in CD14(+) enriched monocyte samples in a blinded fashion. We determined HAND diagnoses by consensus panel and all participants underwent magnetic resonance spectroscopy (MRS) to measure markers of brain injury. Immune activation was measured via cytokines in cerebrospinal fluid (CSF)., Results: The mean (SD) age was 35 (6.9) years, CD4 T-lymphocyte count was 236 (139) and log10 plasma HIV RNA was 4.8 (0.73). Twenty-eight of 61 met HAND criteria. The log10 CD14(+) HIV DNA was associated with HAND in unadjusted and adjusted models (p = 0.001). There was a 14.5 increased odds ratio for HAND per 1 log-value of HIV DNA (10-fold increase in copy number). Plasma CD14(+) HIV DNA was associated with plasma and CSF neopterin (p = 0.023) and with MRS markers of neuronal injury (lower N-acetyl aspartate) and glial dysfunction (higher myoinositol) in multiple brain regions., Interpretation: Reservoir burden of HIV DNA in monocyte-enriched (CD14(+)) peripheral blood cells increases risk for HAND in treatment-naïve HIV+ subjects and is directly associated with CSF immune activation and both brain injury and glial dysfunction by MRS.

Objective: We evaluated regional brain volumes and cerebral metabolite levels as correlates of HIV DNA in peripheral blood mononuclear cells (PBMCs)., Methods: In this cross-sectional study, 35 HIV+ subjects aged ≥40 years (25 with detectable PBMC HIV DNA; 10 with HIV DNA <10 copies/10(6) cells, the threshold of detection) and 12 seronegative controls underwent structural brain MRI and magnetic resonance spectroscopy at 3 T. HIV+ subjects were on combination antiretroviral therapy ≥1 year; all but 1 had plasma HIV RNA <50 copies/mL. We used logistic regression to evaluate relationships of likely predictor variables to the outcome of PBMC HIV DNA detectability in the HIV+ subjects. Effects of serostatus and HIV DNA on regional brain volumes (normalized to intracranial volume) and on metabolite ratios over creatine were evaluated by analyses of covariance, controlling for age., Results: Relative to the HIV+ group with undetectable HIV DNA, subjects with detectable HIV DNA demonstrated decreased volumes of cerebellar (-14%, p = 0.020) and total subcortical (-10%, p = 0.024) gray matter. Compared to healthy controls, only the detectable HIV DNA group showed significant (p < 0.05) enlargement of lateral ventricles and volumetric reductions of caudate, putamen, thalamus, hippocampus, nucleus accumbens, brainstem, total cortical gray matter, and cerebral white matter. Detectable HIV DNA was not associated with significantly altered cerebral metabolite levels., Conclusion: Inability to clear peripheral blood of HIV DNA is associated with regional brain atrophy in well-controlled HIV infection, supporting the involvement of peripheral viral reservoirs in the neuropathogenesis of persistent HIV-related neurocognitive disorders.

Human immunodeficiency virus type 1 (HIV)-infected individuals are at risk for anal cancer, which is caused by human papillomavirus (HPV). The relationship between HIV and HPV that leads to anal cancer remains unclear. Recent data, however, suggest that the continued persistence of HIV DNA in patients treated with combined antiretroviral therapy leads to progression of HIV disease and other HIV-associated complications. Therefore, we investigated the relationship among anal low- and high-grade squamous intraepithelial lesions (LGSIL/HGSIL), high-risk HPV genotypes, and high HIV DNA copy numbers. Anal cytology specimens were assayed for HPV genotype and HIV DNA copy number. High-risk HPV genotypes (odds ratio OR: 3.73; 95% confidence interval CI: 1.08-12.91; p=0.04) and high HIV DNA copy numbers (OR(per 100 HIV DNA copies): 1.13; 95% CI: 1.01-1.27, p=0.04) were both associated with LGSIL/HGSIL. When considering both high-risk HPV genotypes and HIV DNA copy numbers in predicting LGSIL/HGSIL, HIV DNA copy number was significant (OR(per 100 HIV DNA copies): 1.09; 95% CI: 0.96-1.23, p=0.04) but not high-risk HPV genotypes (OR: 2.30, p=0.28), which did not change when adjusted for nadir CD4 cell count and HIV RNA levels. The findings warrant further investigation of HIV DNA and its relationship with HPV in LGSIL/HGSIL pathogenesis.

High levels of human immunodeficiency virus (HIV) DNA in peripheral blood mononuclear cells (PBMCs), and specifically within CD14+ blood monocytes, have been found in HIV-infected individuals with neurocognitive impairment and dementia. The failure of highly active antiretroviral therapy (HAART) to eliminate cognitive dysfunction in HIV may be secondary to persistence of HIV-infected PBMCs which cross the blood-brain barrier, leading to perivascular inflammation and neuronal injury. This study assessed brain cortical thickness relative to HIV DNA levels and identified, we believe for the first time, a neuroimaging correlate of detectable PBMC HIV DNA in subjects with undetectable HIV RNA. Cortical thickness was compared between age- and education-matched groups of older (>40 years) HIV-seropositive subjects on HAART who had detectable (N = 9) and undetectable (N = 10) PBMC HIV DNA. Statistical testing revealed highly significant (P < 0.001) cortical thinning associated with detectable HIV DNA. The largest regions affected were in bilateral insula, orbitofrontal and temporal cortices, right superior frontal cortex, and right caudal anterior cingulate. Cortical thinning correlated significantly with a measure of psychomotor speed. The areas of reduced cortical thickness are key nodes in cognitive and emotional processing networks and may be etiologically important in HIV-related neurological deficits.

HIV-associated neurocognitive disorders (HAND) persist despite plasma HIV RNA suppression with antiretrovirals (ARV). Sequestered reservoirs in the central nervous system and circulating monocytes are theorized to contribute to persistent brain injury. We previously demonstrated that elevated intracellular HIV DNA from circulating cells was associated with HAND in ARV-treated and ARV-naive subjects. We now report that failure to suppress intra-monocyte HIV DNA 3.5 years after initiating ARV is linked to persistent HAND and subjects with dementia are least likely to suppress intra-monocyte HIV DNA at 3.5 years. These findings suggest that antiviral strategies may need to target intra-monocyte HIV DNA.

Human immunodeficiency virus (HIV) DNA in peripheral blood mononuclear cells was previously associated with neuropsychological function. By including individuals encompassing the full range of HIV-1-associated neurocognitive disorders, this study reports results from subjects with normal cognition, minor cognitive motor disorder, and HIV-1-associated dementia. Individuals with normal cognition had relatively low HIV DNA levels compared to those with minor cognitive motor disorder and HIV-1-associated dementia. Neuropsychological deficits were significantly associated with entry HIV DNA in all domains. These findings demonstrate for the first time that the severity of HIV-1-associated neurocognitive disorders is proportional to the amount of circulating HIV DNA.

Objectives: The extent to which highly active antiretroviral therapy (HAART) era cognitive disorders are due to active processes, incomplete clearance of reservoirs, or comorbidities is controversial. This study aimed to determine if immunologic and virologic factors influence cognition after first-time HAART in Thai individuals with HIV-associated dementia (HAD) and Thai individuals without HAD (non-HAD)., Methods: Variables were captured longitudinally to determine factors predictive of degree of cognitive recovery after first-time HAART. Neuropsychological data were compared to those of 230 HIV-negative Thai controls., Results: HIV RNA and CD4 lymphocyte counts were not predictive of HAD cross-sectionally or degree of cognitive improvement longitudinally. In contrast, baseline and longitudinal HIV DNA isolated from monocytes correlated to cognitive performance irrespective of plasma HIV RNA and CD4 lymphocyte counts pre-HAART (p < 0.001) and at 48 weeks post HAART (p < 0.001). Levels exceeding 3.5 log(10) copies HIV DNA/10(6) monocyte at baseline distinguished all HAD and non-HAD cases (p < 0.001). At 48 weeks, monocyte HIV DNA was below the level of detection of our assay (10 copies/10(6) cells) in 15/15 non-HAD compared to only 4/12 HAD cases, despite undetectable plasma HIV RNA in 26/27 cases. Baseline monocyte HIV DNA predicted 48-week cognitive performance on a composite score, independently of concurrent monocyte HIV DNA and CD4 count (p < 0.001)., Conclusions: Monocyte HIV DNA level correlates to cognitive performance before highly active antiretroviral therapy (HAART) and 48 weeks after HAART in this cohort and baseline monocyte HIV DNA may predict 48-week cognitive performance. These findings raise the possibility that short-term incomplete cognitive recovery with HAART may represent an active process related to this peripheral reservoir.

The authors previously found a strong association between elevated HIV proviral DNA (HIV DNA) and a diagnosis of HIV-1-associated dementia (HAD) vs. normal cognition. It is unclear whether HIV DNA globally affects the diagnosis of HAD or whether the effect is limited to individual neuropsychological deficits. This exploratory study examined baseline HIV DNA and its association with individual neuropsychological deficits. HIV DNA was significantly associated with baseline neuropsychological deficits independent of age, ethnicity, IQ, and plasma HIV-1 RNA levels. However, HIV DNA did not predict future changes in neuropsychological deficits. The data suggest that HIV DNA and neuropsychological deficits may co-vary over time.

High HIV-1 DNA (HIV DNA) levels in peripheral blood mononuclear cells (PBMC) correlate with HIV-1-associated dementia (HAD) in patients on highly active antiretroviral therapy (HAART). If this relationship also exists among HAART-naïve patients, then HIV DNA may be implicated in the pathogenesis of HAD. In this study, we evaluated the relationship between HIV DNA and cognition in subjects naïve to HAART in a neuro AIDS cohort in Bangkok, Thailand. Subjects with and without HAD were recruited and matched for age, gender, education, and CD4 cell count. PBMC and cellular subsets were analyzed for HIV DNA using real-time PCR. The median log(10) HIV DNA copies per 10(6) PBMC for subjects with HAD (n=15) was 4.27, which was higher than that found in subjects without dementia (ND; n=15), 2.28, p<0.001. This finding was unchanged in a multivariate model adjusting for plasma HIV-1 RNA levels. From a small subset of individuals, in which adequate number of cells were available, more HIV DNA was in monocytes/macrophages from those with HAD compared to those with ND. These results are consistent with a previous report among HAART-experienced subjects, thus further implicating HIV DNA in the pathogenesis of HAD.

The objective of the study was to assess the feasibility of measuring human immunodeficiency virus 1 (HIV-1) proviral deoxyribonucleic acid (DNA) and associated single-nucleotide polymorphism (SNP) of monocyte chemoattractant protein 1 (MCP1) in pediatric cerebrospinal fluid (CSF). The importance of HIV DNA and MCP1 SNP has been suggested to be independently important in progression to acquired immune deficiency syndrome (AIDS) and neurocognitive impairment in adults. In children, measuring both factors in the CSF may help us understand the neuropathogenic process leading to HIV-1-associated encephalopathy (HAE). Repository specimens from 27 perinatally HIV-1-infected children with HAE were assessed for HIV DNA copy by real-time polymerase chain reaction and compared with MCP1 2578G SNP mutations measured by digesting amplified 361 bp fragments. When compared with MCP1 2578G SNP, a significant number with the mutation had high HIV DNA compared with those with wild type (p < .01), with no levels detected in HIV-1-seronegative control specimens. There were six CSF specimens with enough supernatant to measure MCP1 levels by enzyme-linked immunosorbent assay, which showed high levels in those with the MCP1 2578G mutation. This study demonstrates, for the first time, that CSF HIV DNA and MCP1 SNP can be measured and could be potential tools in future clinical studies to understand the pathogenesis of pediatric HAE.

Objective: Individuals continue to develop HIV-1-associated dementia (HAD) despite treatment with highly active antiretroviral therapy (HAART). Monocytes/macrophages (M/MPhi) can harbor proviral DNA that is not eradicated by HAART. To determine if HAD is associated with the level of HIV-1 infection within circulating leukocytes, we quantified HIV-1 DNA copy number in peripheral blood mononuclear cells (PBMC), and in PBMC subsets., Design: Cross-sectional analysis within the Hawaii Aging with HIV Cohort comparing participants with HAD to those with normal cognition (NC)., Methods: Real-time PCR assays assessing HIV DNA copy number/1 x 10 cells were performed on PBMC and subsets., Results: Individuals with HAD (n = 27) had a median (interquartile range) of 9.11 (37.20) HIV DNA per 1 x 10 PBMC compared to 0.49 (0.89) HIV DNA per 1 x 10 PBMC in individuals with NC (n = 22). Using a univariate analysis in the subset of individuals with undetectable viral load (HAD, n = 11; NC, n = 13), the odds of HAD attributable to HIV DNA copy number was 2.76 (1.28-5.94), P < 0.01. Preliminary analysis of a small subset of patients (n = 5) suggested that the primary source of HIV DNA may be the activated M/MPhi (CD14/CD16) subset., Conclusions: These findings suggest a potentially important association between circulating provirus and HAD.

Measurements of HIV-1 DNA and plasma RNA levels represent unique entities, thus clinically and molecularly, data obtained from each can be used independently in assessing therapy or experiments. Plasma HIV-1 RNA levels are used to make clinical decisions regarding treatment strategies, but viral DNA can still be detectable when plasma RNA levels are undetectable. At the molecular level, accurate assessment of HIV-1 DNA copies/cell could increase the ability to target specific tissues for further analysis such as identification of site-specific integration of HIV in cellular DNA. Using real-time polymerase chain reaction (PCR), HIV-1 copies/cell were determined in peripheral blood mononuclear cells (PBMC), bone marrow (BM), and tissue. Duplicate specimens were analyzed for plasma HIV-1 RNA levels and for viral DNA copies/cell from 24 HIV-1 infected individuals. DNA from an additional 58 PBMC and 34 other tissue specimens were also assayed with the results reported as a log of HIV-1 DNA copies/cell. The log viral DNA copies/cell of the 24 matched specimens ranged from -2.699 to 0.278 with no correlation to the plasma HIV-1 RNA levels (range 52 to 2 X 105 copies/mL). Similar range in log HIV-1 DNA copies/cell was found in the other specimens. Real-time PCR assay for viral DNA copies/cell provides a rapid assessment of HIV-1 copies/cell in specimens independent of plasma HIV-1 RNA levels. From selected cases with relatively high HIV-1 DNA copies/cell, inverse PCR successfully identified viral integration. This type of assay could facilitate further studies when relatively high viral copies/cell are needed for screening.

An essential component of the HIV-1 life cycle involves insertion in the genome of an infected cell. The site of HIV-1 integration has the potential to disrupt a gene and perturb a normal cellular function. To begin to address whether disease pathogenesis may correlate with the site of insertion, flanking cellular sequences at these HIV integrated regions were directly amplified from peripheral blood mononuclear cells DNA from a broad range of infected individuals using an inverse polymerase chain reaction strategy. Amplified flanking regions were sequenced and examined for similarity to the nucleic acid database. In this group of analyzed samples, the HIV-1 provirus was inserted within non-coding regions throughout the genome of the infected host, in which 7/14 sites were positioned in close proximity to different Alu repetitive elements while 2/14 sites were located within intron sequences. Insertions were also detected at sites without a specific gene designation but not within short tandem repetitive sequences, telomeres or centromeric repeat regions. Altogether, it is expected that this approach will yield new information on sites of integration by HIV-1 that may be associated with the pathogenic manifestations of disease progression.

Background: Human herpesvirus-6 (HHV6) has been implicated in adult lymphomas but the role that HHV6 has in lymphomagenesis is unclear. Because primary infection occurs in children, a prospective study was undertaken to detect HHV6 in those with pediatric Hodgkin disease (HD)., Observations: Tumor was obtained from children with HD entered on a Children's Cancer Group Biology Study. Polymerase chain reaction (PCR) was performed using HHV6 primers. All 47 specimens (40 nodular sclerosing; 3 lymphocyte predominant; 3 mixed cellularity; and 1 unclassified) were negative for HHV6., Conclusions: The results demonstrate no HHV6 sequence in childhood HD.