Plasma metabolomics identifies lipid abnormalities linked to markers of inflammation, microbial translocation, and hepatic function in HIV patients receiving protease inhibitors.

BACKGROUND: Metabolic abnormalities are common in HIV-infected individuals on antiretroviral therapy (ART), but the biochemical details and underlying mechanisms of these disorders have not been defined. METHODS: Untargeted metabolomic profiling of plasma was performed for 32 HIV patients with low nadir CD4 counts (<300 cells/ul) on protease inhibitor (PI)-based ART and 20 healthy controls using liquid or gas chromatography and mass spectrometry. Effects of Hepatitis C (HCV) co-infection and relationships between altered lipid metabolites and markers of inflammation, microbial translocation, and hepatic function were examined. Unsupervised hierarchical clustering, principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), Random forest, pathway mapping, and metabolite set enrichment analysis (MSEA) were performed using dChip, Metaboanalyst, and MSEA software. RESULTS: A 35-metabolite signature mapping to lipid, amino acid, and nucleotide metabolism distinguished HIV patients with advanced disease on PI-based ART from controls regardless of HCV serostatus (p<0.05, false discovery rate (FDR)<0.1). Many altered lipids, including bile acids, sulfated steroids, polyunsaturated fatty acids, and eicosanoids, were ligands of nuclear receptors that regulate metabolism and inflammation. Distinct clusters of altered lipids correlated with markers of inflammation (interferon-α and interleukin-6), microbial translocation (lipopolysaccharide (LPS) and LPS-binding protein), and hepatic function (bilirubin) (p<0.05). Lipid alterations showed substantial overlap with those reported in non-alcoholic fatty liver disease (NALFD). Increased bile acids were associated with noninvasive markers of hepatic fibrosis (FIB-4, APRI, and YKL-40) and correlated with acylcarnitines, a marker of mitochondrial dysfunction. CONCLUSIONS: Lipid alterations in HIV patients receiving PI-based ART are linked to markers of inflammation, microbial translocation, and hepatic function, suggesting that therapeutic strategies attenuating dysregulated innate immune activation and hepatic dysfunction may be beneficial for prevention and treatment of metabolic disorders in HIV patients.

Monocyte activation markers in cerebrospinal fluid associated with impaired neurocognitive testing in advanced HIV infection.

BACKGROUND: Activated monocytes/macrophages play a role in severe forms of HIV-associated neurocognitive disorders (HAND), but little is known about the mechanisms driving milder forms that are prevalent despite combination antiretroviral therapy (cART). To examine relationships of monocyte activation markers to HAND of varying severity, we compared plasma and cerebrospinal fluid (CSF) biomarker levels with neurocognitive test scores in HIV+ subjects. METHODS: Plasma and CSF soluble CD14 (sCD14), CCL2, and interleukin (IL) 6 were measured by enzyme-linked immunosorbent assay in 67 HIV+ subjects with nadir CD4 <300, and CSF inflammatory biomarkers were measured by multiplex assay in 14 subjects on suppressive cART. RESULTS: Eighty-two percent were on cART, with 31% having undetectable plasma viral load (VL). CSF sCD14 was increased in subjects with impaired neurocognitive testing (P = 0.02), correlated inversely with global T scores in subjects with detectable but not undetectable plasma VL (P = 0.02), and yielded higher area under the receiver operating characteristic curve values for predicting impaired T scores (0.659) than plasma or CSF VL and current or nadir CD4 counts in single-marker and multivariate models. CSF sCD14, IL-6, IL-8, CCL2, CCL3, CXCL10, and interferon (IFN) gamma were increased in subjects on suppressive cART regardless of cognitive status and predicted patient class in unsupervised analyses, with IL-8, CCL2, and IFNγ explaining most of the variance. CONCLUSIONS: CSF sCD14 is associated with impaired neurocognitive testing in patients with HIV on nonsuppressive cART, suggesting potential utility as a biomarker to monitor HAND progression. CSF sCD14, IL-6, IL-8, CCL2, CCL3, CXCL10, and IFNγ remain elevated in patients on suppressive cART regardless of cognitive status, implying ongoing intrathecal inflammation even in the absence of clinical manifestations.

A plasma biomarker signature of immune activation in HIV patients on antiretroviral therapy.

BACKGROUND: Immune activation is a strong predictor of disease progression in HIV infection. Combinatorial plasma biomarker signatures that represent surrogate markers of immune activation in both viremic and aviremic HIV patients on combination antiretroviral therapy (cART) have not been defined. Here, we identify a plasma inflammatory biomarker signature that distinguishes between both viremic and aviremic HIV patients on cART and healthy controls and examine relationships of this signature to markers of disease progression. METHODS: Multiplex profiling and ELISA were used to detect 15 cytokines/chemokines, soluble IL-2R (sIL-2R), and soluble CD14 (sCD14) in plasma from 57 HIV patients with CD4 nadir <300 cells/µl and 29 healthy controls. Supervised and unsupervised analyses were used to identify biomarkers explaining variance between groups defined by HIV status or drug abuse. Relationships between biomarkers and disease markers were examined by Spearman correlation. RESULTS: The majority (91%) of HIV subjects were on cART, with 38% having undetectable viral loads (VL). Hierarchical clustering identified a biomarker cluster in plasma consisting of two interferon-stimulated gene products (CXCL9 and CXCL10), T cell activation marker (sIL-2R), and monocyte activation marker (sCD14) that distinguished both viremic and aviremic HIV patients on cART from controls (p<0.0001) and were top-ranked in variables important in projection plots. IL-12 and CCL4 were also elevated in viremic and aviremic patients compared to controls (p<0.05). IL-12 correlated with IFNα, IFNγ, CXCL9, and sIL-2R (p<0.05). CXCL10 correlated positively with plasma VL and percentage of CD16+ monocytes, and inversely with CD4 count (p = 0.001, <0.0001, and 0.04, respectively). CONCLUSION: A plasma inflammatory biomarker signature consisting of CXCL9, CXCL10, sIL-2R, and sCD14 may be useful as a surrogate marker to monitor immune activation in both viremic and aviremic HIV patients on cART during disease progression and therapeutic responses.

Plasma sCD14 is a biomarker associated with impaired neurocognitive test performance in attention and learning domains in HIV infection.

OBJECTIVE: Mild forms of HIV-associated neurocognitive disorders (HAND) remain prevalent in the era of combination antiretroviral therapy (cART). Although elevated lipopolysaccharide (LPS) and immune activation are implicated in HAND pathogenesis, relationships of LPS and inflammatory markers to mild forms of HAND or impairment in specific cognitive domains are unknown. To examine these relationships, we compared plasma soluble CD14 (sCD14), CCL2, and LPS levels with neurocognitive test scores in a cART era cohort. METHODS: We analyzed plasma from HIV+ subjects (n = 97) with nadir CD4 counts <300 and high frequency of hepatitis C virus coinfection and illicit drug use for relationships between sCD14, CCL2, and LPS levels and neurocognitive test scores. RESULTS: Plasma sCD14 levels were higher in subjects with test scores indicating global impairment (P = 0.007), particularly in attention and learning domains (P = 0.015 and P = 0.03, respectively), regardless of HAND diagnosis. Plasma sCD14 levels correlated inversely with global, attention, and learning T scores (P = 0.036, 0.047, and 0.007, respectively) and yielded higher area under receiver operating characteristic values for predicting impaired scores than single-marker models based on plasma or cerebrospinal fluid viral load or CD4 count (area under receiver operating characteristic values = 0.71, 0.81, and 0.71, respectively) and in 4-marker models based on plasma sCD14 and 3 conventional markers compared with the 3-marker models. CONCLUSIONS: Plasma sCD14 is a biomarker associated with impaired neurocognitive testing in attention and learning domains in HIV-infected individuals with advanced disease, suggesting involvement of cortical and limbic pathways by inflammatory processes in the cART era. Plasma sCD14 is a potential biomarker to monitor HAND progression and therapeutic responses.

Serological markers for inflammatory bowel disease in AIDS patients with evidence of microbial translocation.

BACKGROUND: Breakdown of the gut mucosal barrier during chronic HIV infection allows translocation of bacterial products such as lipopolysaccharides (LPS) from the gut into the circulation. Microbial translocation also occurs in inflammatory bowel disease (IBD). IBD serological markers are useful in the diagnosis of IBD and to differentiate between Crohn's disease (CD) and ulcerative colitis (UC). Here, we evaluate detection of IBD serological markers in HIV-infected patients with advanced disease and their relationship to HIV disease markers. METHODS: IBD serological markers (ASCA, pANCA, anti-OmpC, and anti-CBir1) were measured by ELISA in plasma from AIDS patients (n = 26) with low CD4 counts (<300 cells/µl) and high plasma LPS levels, and results correlated with clinical data. For meta-analysis, relevant data were abstracted from 20 articles. RESULTS: IBD serological markers were detected in approximately 65% of AIDS patients with evidence of microbial translocation. An antibody pattern consistent with IBD was detected in 46%; of these, 75% had a CD-like pattern. Meta-analysis of data from 20 published studies on IBD serological markers in CD, UC, and non-IBD control subjects indicated that IBD serological markers are detected more frequently in AIDS patients than in non-IBD disease controls and healthy controls, but less frequently than in CD patients. There was no association between IBD serological markers and HIV disease markers (plasma viral load and CD4 counts) in the study cohort. CONCLUSIONS: IBD serological markers may provide a non-invasive approach to monitor HIV-related inflammatory gut disease. Further studies to investigate their clinical significance in HIV-infected individuals are warranted.

Human immunodeficiency virus type 1 V1-to-V5 envelope variants from the chronic phase of infection use CCR5 and fuse more efficiently than those from early after infection.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein modifications over the course of infection have been associated with coreceptor switching and antibody neutralization resistance, but the effect of the changes on replication and host cell receptor usage remains unclear. To examine this question, unique early- and chronic-stage infection envelope V1-to V5 (V1-V5) segments from eight HIV-1 subtype A-infected subjects were incorporated into an isogenic background to construct replication-competent recombinant viruses. In all subjects, viruses with chronic-infection V1-V5 segments showed greater replication capacity than those with early-infection V1-V5 domains in cell lines with high levels of both the CD4 and the CCR5 receptors. Viruses with chronic-infection V1-V5s demonstrated a significantly increased ability to replicate in cells with low CCR5 receptor levels and greater resistance to CCR5 receptor and fusion inhibitors compared to those with early-infection V1-V5 segments. These properties were associated with sequence changes in the envelope V1-V3 segments. Viruses with the envelope segments from the two infection time points showed no significant difference in their ability to infect cells with low CD4 receptor densities, in their sensitivity to soluble CD4, or in their replication capacity in monocyte-derived macrophages. Our results suggest that envelope changes, primarily in the V1-V3 domains, increase both the ability to use the CCR5 receptor and fusion kinetics. Thus, envelope modifications over time within a host potentially enhance replication capacity.

Estimation of virological and immunological parameters in subjects from South India infected with human immunodeficiency virus type 1 clade C and correlation of findings with occurrence of neurological disease.

Several studies carried out in Western countries have demonstrated that a number of virological and immunological markers such as viral loads, cytokines, beta(2)-microglobulin, neopterin, etc., are elevated in the serum and cerebrospinal fluid (CSF) of human immunodeficiency virus (HIV)-infected individuals with neurological disease. The neurological manifestations of HIV infection noted in Indian patients is different from those reported in Western countries. Moreover, few studies have investigated the role of virological and immunological parameters with respect to the progression of HIV-1 clade C infection in India. In this study, we measured virological (HIV-1 RNA levels) and immunological parameters (CD4 cell count and inflammatory markers) in the plasma and CSF of HIV-1-infected neurologically asymptomatic and symptomatic (with opportunistic infections and/or dementia) subjects. By using clade-specific polymerase chain reaction (PCR), we ascertained that all samples used for the study were infected with HIV-1 clade C. Among the various laboratory parameters evaluated, high viral loads in the CSF, low CD4 counts, and higher levels of interleukin (IL)-1alpha, IL-6, tumor necrosis factor alpha (TNFalpha), beta(2)-microglobulin, and neopterin were noted in HIV-infected subjects with neurological disease as compared to asymptomatic subjects. These data suggest that the markers evaluated in plasma and CSF samples correlated with occurrence of neurological disease in symptomatic individuals as compared to asymptomatic HIV infected subjects.

Cognitive changes in asymptomatic drug-naïve human immunodeficiency virus type 1 clade C infection.

Asymptomatic human immunodeficiency virus (HIV) infection is associated with impaired cognitive functioning in both clade B and C infections. The nature of cognitive change longitudinally has not been studied in asymptomatic clade C infection. The present study evaluated changes in neuropsychological functioning over a 2(1/2)-year period in a cohort of HIV-1 clade C-infected asymptomatic individuals from South India. Participants with CD4 counts below 250 were started on highly active antiretroviral therapy (HAART) as per National AIDS Control Organisation NACO guidelines and hence excluded. The sample consisted of 68 patients (30 men and 38 women), with a mean age of 29.4 years (SD=5.6 years) and a mean education of 10.0 years (SD=2.7 years). A comprehensive neuropsychological assessment with 12 tests yielding 21 variables was used to examine cognitive functioning at baseline and subsequently at 6-monthly intervals for five follow-ups. Shift in CD4 and viral load categories measured by the McNemar's test indicated disease progression. Latent growth curve (LGC) modeling assessed the nature of change in cognition over the 2(1/2)-year study period. Ten variables representing attention, executive functions, and long-term memory fit the LGC model. Excepting visual working memory, the slope was nonsignificant for nine variables, indicating absence of deterioration in cognition over a 2(1/2)-year period. However, CD4 and viral load levels worsened, indicating disease progression. Asymptomatic individuals with HIV-1 clade C infection do not show any significant decline on individual neuropsychological functions over 2(1/2) years despite disease progression, as evidenced by immune suppression and viral loads.

Microbial translocation is associated with increased monocyte activation and dementia in AIDS patients.

Elevated plasma lipopolysaccharide (LPS), an indicator of microbial translocation from the gut, is a likely cause of systemic immune activation in chronic HIV infection. LPS induces monocyte activation and trafficking into brain, which are key mechanisms in the pathogenesis of HIV-associated dementia (HAD). To determine whether high LPS levels are associated with increased monocyte activation and HAD, we obtained peripheral blood samples from AIDS patients and examined plasma LPS by Limulus amebocyte lysate (LAL) assay, peripheral blood monocytes by FACS, and soluble markers of monocyte activation by ELISA. Purified monocytes were isolated by FACS sorting, and HIV DNA and RNA levels were quantified by real time PCR. Circulating monocytes expressed high levels of the activation markers CD69 and HLA-DR, and harbored low levels of HIV compared to CD4(+) T-cells. High plasma LPS levels were associated with increased plasma sCD14 and LPS-binding protein (LBP) levels, and low endotoxin core antibody levels. LPS levels were higher in HAD patients compared to control groups, and were associated with HAD independently of plasma viral load and CD4 counts. LPS levels were higher in AIDS patients using intravenous heroin and/or ethanol, or with Hepatitis C virus (HCV) co-infection, compared to control groups. These results suggest a role for elevated LPS levels in driving monocyte activation in AIDS, thereby contributing to the pathogenesis of HAD, and provide evidence that cofactors linked to substance abuse and HCV co-infection influence these processes.

Neuropsychological deficits in human immunodeficiency virus type 1 clade C-seropositive adults from South India.

Most studies of cognitive functioning in human immunodeficiency virus type 1 (HIV-1)-seropositive (HIV-1+) subjects have been done in the United States and Europe, where clade B infections predominate. However, in other parts of the world such as South India, where clade C HIV is most common, the prevalence of HIV-1 is increasing. Standardized neuropsychological tests were used to assess cognitive functioning in a sample of 119 adults infected with clade C HIV-1 who were not on antiretroviral medications. The subjects did not have neurological or psychiatric illness and were functioning adequately. Neuropsychological test performance was compared with gender-, age-, and education-matched normative data derived from a sample of 540 healthy volunteers and a matched cohort of 126 healthy, HIV-1-seronegative individuals. Among the seropositive subjects, 60.5% had mild to moderate cognitive deficits characterized by deficits in the domains of fluency, working memory, and learning and memory. None of the subjects had severe cognitive deficits. The HIV-1+ sample was classified into groups according to the level of immune suppression as defined by CD4 count (< 200, 201-499, and > 500 cells/mm3) and viral load (< 5000, 5001-30,000, 30,001-99,999, 100,000-1,000,000, and > 1,000,001 copies). Although the most immunosuppressed group (CD4 count < 200 cells/mm3 or viral load > 1,000,001 copies) was small, their rate of impairment in visual working memory was greater when compared to groups with better immune functioning. Mild to moderate cognitive deficits can be identified on standardized neuropsychological tests in clade C-infected HIV-1+ adults who do not have any clinically identifiable functional impairment. The prevalence of cognitive deficits is similar to that reported in antiretroviral treatment-naïve individuals infected with clade B virus in the western world.

Quantitation of HIV-1 RNA levels in plasma and CSF of asymptomatic HIV-1 infected patients from South India using a TaqMan real time PCR assay.

BACKGROUND: Most of the quantitation assays for HIV-1 RNA used currently are designed and optimized for HIV-1 subtype B viruses and hence may not be suitable for India, where the predominant subtype is HIV-1 subtype C. OBJECTIVES: Development and standardization of HIV-1 TaqMan real time PCR assay suitable for measuring plasma and CSF viral RNA levels in HIV subtype C infected individuals. STUDY DESIGN: A TaqMan real time PCR was developed using primers and probes selected in the gag region for detection of Indian HIV-1 subtype C strain. Plasma (n=120) and CSF samples (n=46) obtained from HIV infected subjects were used to evaluate the sensitivity and specificity of the assay. A comparative evaluation was carried out with a commercially available quantitative HIV viral load assay (Roche Amplicor Version 1.5). RESULTS: The TaqMan assay was able to amplify all HIV-1 group M subtypes except subtype E. Viral loads could be estimated in all the plasma (n=120) and 40/46 CSF samples obtained from HIV positive subjects. Sensitivity of this assay was found to be 180 copies/ml. Correlation with the commercially available viral load assay was very good (r=0.885). CONCLUSIONS: A TaqMan real time PCR was standardized for HIV-1 subtype C and it was more sensitive (180 copies/ml) than standard Amplicor monitor assay, Version 1.5 (400 copies/ml).

Quality of life in HIV subtype C infection among asymptomatic subjects and its association with CD4 counts and viral loads--a study from South India.

OBJECTIVE: To study the association between quality of life (QOL) domains and biological markers of disease progression of HIV infection, i.e. viral load (VL) and CD4 counts among asymptomatic subjects with HIV subtype C infection in South India. DESIGN: Quality of life was measured using the locally validated version of the WHOQOL HIV-BREF. The subjects were neurologically asymptomatic, non psychiatrically ill HIV infected men and women participating in a cohort study. RESULTS: The results indicated mixed findings, with some QOL dimensions being associated with high VLs and low CD4 counts while several others did not show any associations. Significant associations were seen between low CD4 counts and the psychological and social relationships domain, with lower mean scores in these domains being reported by subjects having CD4 counts <200/mm. However, there were no significant differences between the CD4 subgroups for the domains related to physical health, level of independence, environment, and spirituality domains. Significant lower mean QOL scores were found in the highest VL subgroup compared to other groups for the following WHOQOL HIV-BREF domains: physical, psychological, level of independence, and environmental. CONCLUSIONS: In this sample of HAART naïve asymptomatic HIV infected subjects, some QOL dimensions were associated with the biological markers of disease progression i.e. VL and CD4 counts, while several were not. The associations were significant only in the high VL and low CD4 groups.

Ante mortem diagnosis of human rabies using saliva samples: comparison of real time and conventional RT-PCR techniques.

BACKGROUND: Rabies is an enzootic and fatal disease and is still a major problem in developing countries. Ante mortem diagnosis in human cases is necessary for medical management of the patient and to ensure appropriate post-exposure treatment of contacts. Both conventional RT-PCR and Real time PCR (TaqMan) have been described for the detection of rabies virus RNA from saliva and tissue respectively, however to date, there have been no studies comparing conventional and real time PCR assays for detection of rabies virus nucleic acid using saliva samples for ante mortem diagnosis. OBJECTIVES: In this study, we evaluated the utility of conventional RT-PCR and SYBR Green I Real time PCR in the ante mortem diagnosis of rabies using saliva samples. STUDY DESIGN: Saliva samples collected from twenty-four patients presenting with typical clinical manifestations of rabies were tested in the two assays. RESULTS: Amongst the 24 samples tested, 21 samples (87.5%) were positive by either of the two molecular methods. Of these 21, rabies virus RNA was detected in 6/21 in the conventional RT-PCR assay while SYBR Green I Real time PCR could detect RNA in 18/21 samples. CONCLUSION: Real time PCR assay was more sensitive than conventional RT-PCR assay (sensitivity 75% versus 37%, p=0.0189). This study highlights the utility of molecular diagnostic tests in establishing ante mortem diagnosis of rabies using saliva samples within a few hours.