HIV+ Neurocognitive Disorders and Immune Activation
HIV+ Neurocognitive Disorders and Immune Activation
A cross-sectional analysis was performed which included patients from two existing cohorts, those of the Neuradapt and Eldadapt studies, the latter of which is still ongoing, where patients were randomly selected to undergo neuropsychological (NP) evaluation from 2007 to 2013. Both studies received approval from the Ethics Committee of Montpellier, France.
Inclusion criteria in this analysis were: being an adult over 18 years of age with HIV-1 seropositive status, the ability to provide written consent, viral load below 40 HIV-1 RNA copies/ml at inclusion [Abbott real-time polymerase chain reaction (PCR); Abbott real-time HIV-1 2G31–90, Abbott Molecular Inc, De Plaines, IL, USA] and receiving stable treatment (i.e. no change in cART during at least the past 6 months).
Exclusion criteria included active opportunistic infection, a change in psychotropic therapy within the last 3 weeks or any neurological history.
Each patient performed a wide range of NP tests administered by two trained neuropsychologists, one for the Neuradapt study (Nice) and one for the Eldadapt study (Nice and Cannes). They were both highly trained in NP evaluation of HIV-infected patients and stayed in close contact in order to ensure consistency in rigorously following the AAN revised criteria for the definition of HAND.
The tests explored a wide spectrum of cognitive domains: learning and recall episodic memory, attention/concentration, working memory, executive functions, language, visual recognition and motor/psychomotor speed.
The Neuradapt and Eldadapt studies explored the same cognitive domains and strictly followed the AAN revised criteria for defining HAND.
The following NP tests were used in both studies:
For attention/concentration and working memory, the Four Seconds Paced Auditory Serial Addition Task (PASAT) was used in the Neuradapt study, while the Eldadapt study used Digit Span and Letter-Number-Sequencing. In addition, the Motor and Psychomotor Tests (timed finger tapping and timed alternating hand sequence tests) were chosen in the Neuradapt study for motor and psychomotor speed abilities, while Eldadapt used the Grooved Pegboard. Finally, the Modified Card Sorting Test assessed executive functioning in the Neuradapt study whereas the Matrix Reasoning Test and the Trail Making Test were used in Eldadapt.
The NP scores from each test were transformed into z-scores as described elsewhere and were adjusted for age, gender and years of education, using standardized norms.
The mean duration of tests was 90 min per patient. Patients were also assessed according to the Montgomery and Asberg Depression Rating Scale (MADRS) to identify potential behavioural disorders.
According to the AAN revised criteria, patients with HAND can be subdivided into the following three categories.
Patients with HAD were excluded from the analysis because the physiopathology of their brain injury and its clinical consequences are very different from those in subjects with MND. Only subjects with MND were therefore included in the sHAND group.
Thus, each patient performing 1 SD or more below the mean in at least two cognitive domains was considered as presenting with HANDs and classified as having ANI or MND, according to the degree of interference with daily living.
To investigate potential correlations with NP test results, the following parameters were recorded for each patient: age, gender, education, comorbid conditions (hypertension, dyslipidaemia and diabetes), risk factors (smoking, illicit drug use and use of psychotropic molecules, i.e. benzodiazepines, antidepressants, carbamates and anti-epileptic drugs), CD4 and CD8 T-cell counts and CD4:CD8 ratio at inclusion, nadir CD4 T-cell count, time since HIV infection, previous AIDS-defining events, global duration of cART, time on current cART and viral hepatitis markers.
Samples were stained within 8 h of blood draw. CD38 and HLA-DR expression was measured on CD4 and CD8+ T-cells by six-colour flow cytometry using a whole blood cell procedure. Briefly, 50 mL of whole blood was incubated with a mixture of antibodies (all from BD Biosciences, San Jose, CA, USA) namely CD3 fluorescein isothiocyanate (FITC), CD8 peridinin-chrorophyll-protein-cyanin 5.5 (PerCP-Cy5.5), CD4 phycoerythrin cyanin 7 (PC7), CD45 allophycocyanin cyanin 7 (APC-Cy7) and either CD38 phycoerythrin (PE) and anti HLA-Dr allophycocyanin (APC) or their irrelevant counterparts (same fluorochrome-labelled antibody but whose antigen binding sites are directed against a non-human antigen that is keyhole limpet hemocyanin and used to detect nonspecific binding) for 15 min in the dark. The red blood cells were lysed using 2 mL of FACS lysing solution (BD Biosciences), washed before being suspended in 500 mL of Cell Wash (BD Biosciences). A minimum of 10.000 lymphocytes were acquired for each condition. Flow cytometric acquisition and analysis were performed on a FACSCanto flow cytometer and analysis was performed using FACSDiva software (v6.1, BD Biosciences).
First, the CD4:CD8 ratio was correlated as a quantitative variable with NP testing results. Means were calculated using analysis of variance (ANOVA).
In particular, three groups of patients were identified according to NP testing results:
Contrasts were calculated using the Tukey method.
A multinominal logistic regression was then performed with a cut-off at 1 for the CD4:CD8 ratio, as described in previous studies. Variables with P ≤ 0.1 in univariate analysis were initially selected for the multivariate model and only those with P ≤ 0.05 were retained in the final model.
Finally, we analysed factors associated with the CD4:CD8 ratio, in order to exclude potential confounding in its correlation with NP test results.
Methods
Participants
A cross-sectional analysis was performed which included patients from two existing cohorts, those of the Neuradapt and Eldadapt studies, the latter of which is still ongoing, where patients were randomly selected to undergo neuropsychological (NP) evaluation from 2007 to 2013. Both studies received approval from the Ethics Committee of Montpellier, France.
Inclusion criteria in this analysis were: being an adult over 18 years of age with HIV-1 seropositive status, the ability to provide written consent, viral load below 40 HIV-1 RNA copies/ml at inclusion [Abbott real-time polymerase chain reaction (PCR); Abbott real-time HIV-1 2G31–90, Abbott Molecular Inc, De Plaines, IL, USA] and receiving stable treatment (i.e. no change in cART during at least the past 6 months).
Exclusion criteria included active opportunistic infection, a change in psychotropic therapy within the last 3 weeks or any neurological history.
Neuropsychological Analysis
Each patient performed a wide range of NP tests administered by two trained neuropsychologists, one for the Neuradapt study (Nice) and one for the Eldadapt study (Nice and Cannes). They were both highly trained in NP evaluation of HIV-infected patients and stayed in close contact in order to ensure consistency in rigorously following the AAN revised criteria for the definition of HAND.
The tests explored a wide spectrum of cognitive domains: learning and recall episodic memory, attention/concentration, working memory, executive functions, language, visual recognition and motor/psychomotor speed.
The Neuradapt and Eldadapt studies explored the same cognitive domains and strictly followed the AAN revised criteria for defining HAND.
The following NP tests were used in both studies:
the Mini Mental State Evaluation (MMSE) for evaluation of global cognitive function;
the Grober and Buschke test for episodic memory (learning and recall);
the Stroop test for attention/concentration and speed of information processing;
Verbal Fluency for executive functions and language;
Protocole Montreal-Toulouse d'Evaluation des Gnosies Visuelles for visual agnosia.
For attention/concentration and working memory, the Four Seconds Paced Auditory Serial Addition Task (PASAT) was used in the Neuradapt study, while the Eldadapt study used Digit Span and Letter-Number-Sequencing. In addition, the Motor and Psychomotor Tests (timed finger tapping and timed alternating hand sequence tests) were chosen in the Neuradapt study for motor and psychomotor speed abilities, while Eldadapt used the Grooved Pegboard. Finally, the Modified Card Sorting Test assessed executive functioning in the Neuradapt study whereas the Matrix Reasoning Test and the Trail Making Test were used in Eldadapt.
The NP scores from each test were transformed into z-scores as described elsewhere and were adjusted for age, gender and years of education, using standardized norms.
The mean duration of tests was 90 min per patient. Patients were also assessed according to the Montgomery and Asberg Depression Rating Scale (MADRS) to identify potential behavioural disorders.
According to the AAN revised criteria, patients with HAND can be subdivided into the following three categories.
Patients with ANI, involving at least two cognitive domains and demonstrated by a performance of at least 1 standard deviation (SD) below the mean on NP tests, without interference with everyday functioning. The asymptomatic characteristics of impairment were defined using the Instrumental Activity of Daily Living short version battery and by interviewing the patient and his/her family.
Patients with MND, involving at least two cognitive domains and demonstrated by a performance of at least 1 SD below the mean on NP tests, with mild interference with daily functioning.
Patients with HAD, involving at least two cognitive domains and demonstrated by a performance of 2 SD below the normative mean on NP tests, with marked interference with daily functioning.
Patients with HAD were excluded from the analysis because the physiopathology of their brain injury and its clinical consequences are very different from those in subjects with MND. Only subjects with MND were therefore included in the sHAND group.
Thus, each patient performing 1 SD or more below the mean in at least two cognitive domains was considered as presenting with HANDs and classified as having ANI or MND, according to the degree of interference with daily living.
Demographic Parameters and Background Measurements
To investigate potential correlations with NP test results, the following parameters were recorded for each patient: age, gender, education, comorbid conditions (hypertension, dyslipidaemia and diabetes), risk factors (smoking, illicit drug use and use of psychotropic molecules, i.e. benzodiazepines, antidepressants, carbamates and anti-epileptic drugs), CD4 and CD8 T-cell counts and CD4:CD8 ratio at inclusion, nadir CD4 T-cell count, time since HIV infection, previous AIDS-defining events, global duration of cART, time on current cART and viral hepatitis markers.
Measurements of T-cell Activation Markers
Samples were stained within 8 h of blood draw. CD38 and HLA-DR expression was measured on CD4 and CD8+ T-cells by six-colour flow cytometry using a whole blood cell procedure. Briefly, 50 mL of whole blood was incubated with a mixture of antibodies (all from BD Biosciences, San Jose, CA, USA) namely CD3 fluorescein isothiocyanate (FITC), CD8 peridinin-chrorophyll-protein-cyanin 5.5 (PerCP-Cy5.5), CD4 phycoerythrin cyanin 7 (PC7), CD45 allophycocyanin cyanin 7 (APC-Cy7) and either CD38 phycoerythrin (PE) and anti HLA-Dr allophycocyanin (APC) or their irrelevant counterparts (same fluorochrome-labelled antibody but whose antigen binding sites are directed against a non-human antigen that is keyhole limpet hemocyanin and used to detect nonspecific binding) for 15 min in the dark. The red blood cells were lysed using 2 mL of FACS lysing solution (BD Biosciences), washed before being suspended in 500 mL of Cell Wash (BD Biosciences). A minimum of 10.000 lymphocytes were acquired for each condition. Flow cytometric acquisition and analysis were performed on a FACSCanto flow cytometer and analysis was performed using FACSDiva software (v6.1, BD Biosciences).
Statistical Analysis
First, the CD4:CD8 ratio was correlated as a quantitative variable with NP testing results. Means were calculated using analysis of variance (ANOVA).
In particular, three groups of patients were identified according to NP testing results:
unimpaired subjects;
patients with ANI;
patients with sHAND.
Contrasts were calculated using the Tukey method.
A multinominal logistic regression was then performed with a cut-off at 1 for the CD4:CD8 ratio, as described in previous studies. Variables with P ≤ 0.1 in univariate analysis were initially selected for the multivariate model and only those with P ≤ 0.05 were retained in the final model.
Finally, we analysed factors associated with the CD4:CD8 ratio, in order to exclude potential confounding in its correlation with NP test results.
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