Cerebrospinal Fluid Viral Load Research Articles

Cerebrospinal Fluid Viral Escape on Highly Active Antiretroviral Therapy: Analysis from Single Tertiary Care Centre.

HIV-infected individuals receiving regular antiretroviral therapy (ART) can present with a high viral load in cerebrospinal fluid (CSF) at times when it is suppressed in blood. This study presents data of HIV-infected patients who had undetectable or low plasma viral load in blood but presented with neurological signs and symptoms and were diagnosed to have CSF HIV viral escape. Records were reviewed for clinical manifestations, details of opportunistic or coinfection, and HIV viral copies in plasma and CSF at time of diagnosis of CSF escape. A total of 10,200 HIV-infected individuals were registered in HIV care till December 31, 2021. Nineteen individuals (14 virologically confirmed and 5 clinically) were diagnosed with high viral copies in CSF from June 2014 to December 2021. Mean age was 41.5 ± 9.2 (median, 39.5; range, 30-62) years. Average duration of antiretroviral treatment received at the time of diagnosis of CSF escape was 10.1 years. Median plasma HIV-viral copies were 2,469.8 (undetectable to 29,418) and in CSF were 12,773.7 (n = 14, range, 1,340-48,530) copies/mL. HIV viral copies in CSF were significantly higher than in plasma at the time of presentation (p = .003). ART regimen switch was done after identification of HIV CSF escape. Seventeen patients were alive with a regular follow-up of average 35 (range 7-66) months. All had documented clinical improvement with reversal of neurological impairment after ART switch. There was one death and one lost to follow-up. Early identification and timely intervention in CSF viral escape could revert severe neurological impairment and improves treatment outcome.

Evaluation of enterovirus concentration, species identification, and cerebrospinal fluid parameters in patients of different ages with aseptic meningitis in São Paulo, Brazil.

Human enteroviruses (EV) are the most common cause of aseptic meningitis worldwide. Data on EV viral load in cerebrospinal fluid (CSF) and related epidemiological studies are scarce in Brazil. This study investigated the influence of EV viral load on CSF parameters, as well as identifying the involved species. CSF samples were collected in 2018-2019 from 140 individuals at The Hospital das Clínicas, São Paulo. The EV viral load was determined usingreal-time quantitative polymerase chain reaction,while EV species were identified by5'UTR region sequencing.Median viral load was 5.72 log10 copies/mL and did not differ by subjects'age and EV species. Pleocytosis was observed in 94.3% of cases, with the highest white blood cell (WBC) counts in younger individuals. Viral load and WBC count were correlated in children (p = 0.0172). Elevated lactate levels were observed in 60% of cases and correlated with the viral load in preteen-teenagers (p = 0.0120) and adults (p = 0.0184). Most individuals had normal total protein levels (70.7%), with higher in preteen-teenagers and adults (p < 0.0001). By sequencing, 8.2% were identified as EV species A and 91.8% as species B. Age-specific variations in CSF characteristics suggest distinct inflammatory responses in each group.

Detection of human herpesvirus 6 in pediatric CSF samples: causing disease or incidental distraction?

Interpretation of human herpesvirus type 6 (HHV6) detection in the cerebrospinal fluid (CSF) of children can be complex; the virus can cause acute infection, reactivation, or can be inherited chromosomally integrated (iciHHV6). Our objectives were to determine the prevalence of HHV6 including iciHHV6 in CSF and compare the clinical and laboratory characteristics with and without iciHHV6 in our patient population. Overall, the prevalence of HHV6 and iciHHV6 was 2.4% and 0.85%, respectively. Children with iciHHV6 were significantly younger and less likely to present with fever. Septic infants (≤60 days) accounted for 65.2% (15/23) of the iciHHV6 patients. Patients with iciHHV6 had higher viral loads in CSF and whole blood. Twenty-one (91.3%) patients with iciHHV6 and 12 (33.3%) without ici-HHV6 were determined to have an incidental detection of HHV6 not associated with presenting symptoms. Molecular detection of HHV6 in CSF is not always associated with HHV6 infection and may represent iciHHV6 particularly in infants evaluated for sepsis.

Characteristics and Treatment of anti-NMDAR Encephalitis in People Living with HIV: A Case Report and Literature Review (P5-5.005)

<h3>Objective:</h3> To describe the risk factors, symptoms, treatment, and outcomes of anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis occurring in people living with HIV (PLWH). <h3>Background:</h3> Herpes simplex virus encephalitis is a known trigger of anti-NMDAR encephalitis. In recent years, multiple case reports also document anti-NMDAR encephalitis in PLWH, especially those who develop cerebrospinal fluid (CSF) escape, defined by an elevated CSF viral load in the setting of plasma viral suppression. Although the relationship between HIV and other viruses with anti-NMDAR encephalitis has not been fully elucidated, possible mechanisms include molecular mimicry or viral-induced release of NMDAR and other proteins as a result of neurotoxicity. HIV-induced functional dysregulation of CD4+ cell subtypes is another plausible contributor. <h3>Design/Methods:</h3> We conducted a literature search for published cases of autoimmune encephalitis in PLWH. We added an unpublished case from our center and described the clinical features of this unique cohort. <h3>Results:</h3> Eight published cases of anti-NMDAR encephalitis among PLWH were identified, as well as one unpublished case from our center. Common trends in these 9 cases include elevated CSF HIV viral load, T2 hyperintense lesions on MRI, and clinical improvement with immunomodulation. Our patient was a 51-year-old woman who presented off of antiretroviral therapy (recent CD4 range 199–252 cells/mm3) with status epilepticus and was found to have an elevated CSF HIV viral load and multiple non-enhancing T2-hyperintense lesions on brain MRI. Anti-NMDAR antibodies were positive in the serum and CSF. She did not respond to steroids or intravenous immunoglobulins, but began to improve after plasma exchange and was subsequently treated with rituximab. <h3>Conclusions:</h3> Anti-NMDAR encephalitis is an important, treatable consideration in PLWH who present with altered mental status or new seizures. Patients with an elevated CSF viral load may be at higher risk for this syndrome, and immunomodulation may be indicated even in patients who are immunosuppressed. <b>Disclosure:</b> Dr. White has nothing to disclose. Dr. Vila has nothing to disclose. The institution of Dr. Bissonnette has received research support from Michael J Fox Foundation. The institution of Dr. Bissonnette has received research support from Cogan Family Foundation. Dr. Martinez-Ramirez has nothing to disclose. Dr. Anand has nothing to disclose. Dr. Cervantes-Arslanian has nothing to disclose.

Rebound HIV-1 in cerebrospinal fluid after antiviral therapy interruption is mainly clonally amplified R5 T cell-tropic virus.

HIV-1 persists as a latent reservoir in people receiving suppressive antiretroviral therapy (ART). When ART is interrupted (treatment interruption/TI), rebound virus re-initiates systemic infection in the lymphoid system. During TI, HIV-1 is also detected in cerebrospinal fluid (CSF), although the source of this rebound virus is unknown. To investigate whether there is a distinct HIV-1 reservoir in the central nervous system (CNS), we compared rebound virus after TI in the blood and CSF of 11 participants. Peak rebound CSF viral loads vary and we show that high viral loads and the appearance of clonally amplified viral lineages in the CSF are correlated with the transient influx of white blood cells. We found no evidence of rebound macrophage-tropic virus in the CSF, even in one individual who had macrophage-tropic HIV-1 in the CSF pre-therapy. We propose a model in which R5 T cell-tropic virus is released from infected T cells that enter the CNS from the blood (or are resident in the CNS during therapy), with clonal amplification of infected T cells and virus replication occurring in the CNS during TI.

Longitudinal white matter alterations in SIVmac239-infected rhesus monkeys with and without regular cART treatment.

To use SIV-mac239-infected Chinese rhesus monkeys to study white matter changes with and without regular combined antiretroviral therapy (cART) and the relationships between the changes and clinical results. Diffusion tensor imaging (DTI) data were collected at baseline and 10 days, 4 weeks, 12 weeks, 24 weeks, and 36 weeks after viral inoculation. Plasma CD4 T cell counts, CD4/CD8 ratio, plasma viral load, and cerebrospinal fluid (CSF) viral load were collected at baseline and 1 week, 5 weeks, 12 weeks, 24 weeks, and 36 weeks after viral inoculation. Microstructural characteristics were examined within 76 white matter areas defined by the DTI-white matter (WM) atlas for rhesus macaques. Corrections for multiple comparisons were performed using a false discovery rate (p < 0.05, FDR). Correlation analyzes between imaging markers and clinical markers (plasma CD4 T cell counts, CD4/CD8 ratio, plasma viral load, and cerebral spinal fluid viral load) were performed using Pearson correlations. White matter changes in SIV-infected macaques were detected in different brain regions as early as 4 weeks after inoculation. As time progressed, cART reversed, ameliorated, or even enhanced the effects. The CD4 T cell count was mainly associated with DTI metrics before cART, while the CD4/CD8 ratio was associated with white matter changes with and without cART. Viral load was positively associated with mean diffusivity in HIV patients without cART, and the opposite results were seen in HIV patients with cART. SIV-mac239 infection may be an ideal tool for studying HIV-induced changes in the brain. The first white matter changes appeared in a structure adjacent to the periventricular area as early as 4 weeks after inoculation. As time progressed, cART had different effects on different regions, reversing, attenuating, or even progressing the pathology. Moreover, these changes were closely related to the CD4/CD8 ratio and viral load, even after cART.

Opsoclonus Myoclonus Ataxia Syndrome in a HIV Patient as a rare presentation of CSF escape: A Case Report

Cerebrospinal fluid escape is an uncommon phenomenon seen in patients where there is discordance in plasma and cerebrospinal fluid viral load. The presentation of this condition can range from being asymptomatic to the patient showing cognitive dysfunction. In addition, it can also be associated with various opportunistic neurological infections. Opsoclonus Myoclonus Ataxia is very rare entity classically associated with neuroblastoma or viral illness. Opsoclonus Myoclonus Ataxia Syndrome (OMAS) has previously been described as a presentation of Human Immunodeficiency Virus cerebrospinal fluid escape very rarely. We report a case of 55 years old patient diagnosed with HIV/AIDS, who was on first line antiretroviral therapy and presented with Opsoclonus Myoclonus Ataxia Syndrome associated with Cerebral Fluid (CSF) Escape, along with the review of the literature. Moreover, in this article, we're providing a postulation for the plausible cause behind cerebrospinal fluid escape leading to the Opsoclonus Myoclonus Ataxia Syndrome. Keywords: HIV/AIDS, CSF Escape, Opsoclonus Myoclonus Ataxia Syndrome.

Reversal of CSF HIV-1 Escape during Treatment of HIV-Associated Cryptococcal Meningitis in Botswana.

Cerebrospinal fluid (CSF) viral escape has been poorly described among people with HIV-associated cryptococcal meningitis. We determined the prevalence of CSF viral escape and HIV-1 viral load (VL) trajectories in individuals treated for HIV-associated cryptococcal meningitis. A retrospective longitudinal study was performed using paired CSF and plasma collected prior to and during the antifungal treatment of 83 participants recruited at the Botswana site of the phase-3 AMBITION-cm trial (2018–2021). HIV-1 RNA levels were quantified then CSF viral escape (CSF HIV-1 RNA ≥ 0.5 log10 higher than plasma) and HIV-1 VL trajectories were assessed. CSF viral escape occurred in 20/62 (32.3%; 95% confidence interval [CI]: 21.9–44.6%), 13/52 (25.0%; 95% CI: 15.2–38.2%) and 1/33 (3.0%; 95% CI: 0.16–15.3%) participants at days 1, 7 and 14 respectively. CSF viral escape was significantly lower on day 14 compared to days 1 and 7, p = 0.003 and p = 0.02, respectively. HIV-1 VL decreased significantly from day 1 to day 14 post antifungal therapy in the CSF but not in the plasma (β = −0.47; 95% CI: −0.69 to −0.25; p < 0.001). CSF viral escape is high among individuals presenting with HIV-associated cryptococcal meningitis; however, antifungal therapy may reverse this, highlighting the importance of rapid initiation of antifungal therapy in these patients.

Cerebrospinal Fluid Viral Load Across the Spectrum of Untreated Human Immunodeficiency Virus Type 1 (HIV-1) Infection: A Cross-Sectional Multicenter Study.

BackgroundThe aim of this large multicenter study was to determine variations in cerebrospinal fluid (CSF) HIV-RNA in different phases of untreated human immunodeficiency virus type 1 (HIV-1) infection and its associations with plasma HIV-RNA and other biomarkers.MethodsTreatment naive adults with available CSF HIV-RNA quantification were included and divided into groups representing significant disease phases. Plasma HIV-RNA, CSF white blood cell count (WBC), neopterin, and albumin ratio were included when available.ResultsIn total, 1018 patients were included. CSF HIV-RNA was in median (interquartile range [IQR]) 1.03 log10 (0.37–1.86) copies/mL lower than in plasma, and correlated with plasma HIV-RNA (r = 0.44, P < .01), neopterin concentration in CSF (r = 0.49, P < .01) and in serum (r = 0.29, P < .01), CSF WBC (r = 0.34, P < .01) and albumin ratio (r = 0.25, P < .01). CSF HIV-RNA paralleled plasma HIV-RNA in all groups except neuroasymptomatic patients with advanced immunodeficiency (CD4 < 200) and patients with HIV-associated dementia (HAD) or opportunistic central nervous system (CNS) infections. Patients with HAD had the highest CSF HIV-RNA (in median [IQR] 4.73 (3.84–5.35) log10 copies/mL). CSF > plasma discordance was found in 126 of 972 individuals (13%) and varied between groups, from 1% in primary HIV, 11% in neuroasymptomatic groups, up to 30% of patients with HAD.ConclusionsOur study confirms previous smaller observations of variations in CSF HIV-RNA in different stages of HIV disease. Overall, CSF HIV-RNA was approximately 1 log10 copies/mL lower in CSF than in plasma, but CSF discordance was found in a substantial minority of subjects, most commonly in patients with HAD, indicating increasing CNS compartmentalization paralleling disease progression.

Adoptive Transfer of JC Virus-Specific T Lymphocytes for the Treatment of Progressive Multifocal Leukoencephalopathy.

ObjectiveProgressive multifocal leukoencephalopathy (PML) is still burdened by high mortality in a subset of patients, such as those affected by hematological malignancies. The aim of this study was to analyze the safety and carry out preliminary evaluation of the efficacy of polyomavirus JC (JCPyV)‐specific T cell therapy in a cohort of hematological patients with PML.MethodsBetween 2014 and 2019, 9 patients with a diagnosis of “definite PML” according to the 2013 consensus who were showing progressive clinical deterioration received JCPyV‐specific T cells. Cell lines were expanded from autologous or allogenic peripheral blood mononuclear cells by stimulation with JCPyV antigen‐derived peptides.ResultsNone of the patients experienced treatment‐related adverse events. In the evaluable patients, an increase in the frequency of circulating JCPyV‐specific lymphocytes was observed, with a decrease or clearance of JCPyV viral load in cerebrospinal fluid. In responsive patients, transient appearance of punctate areas of contrast enhancement within, or close to, PML lesions was observed, which was interpreted as a sign of immune control and which regressed spontaneously without the need for steroid treatment. Six of 9 patients achieved PML control, with 5 alive and in good clinical condition at their last follow‐up.InterpretationAmong other novel treatments, T cell therapy is emerging as a viable treatment option in patients with PML, particularly for those not amenable to restoration of specific immunity. Neurologists should be encouraged to refer PML patients to specialized centers to allow access to this treatment strategy. ANN NEUROL 2021;89:769–779

Inadequate Immune Humoral Response against JC Virus in Progressive Multifocal Leukoencephalopathy Non-Survivors.

JC virus (JCV) causes progressive multifocal leukoencephalopathy (PML) in immunosuppressed patients. There is currently no effective specific antiviral treatment and PML management relies on immune restoration. Prognosis markers are crucially needed in this disease because of its high mortality rate. In this work, we investigated the compartmentalization of JCV strains as well as the humoral neutralizing response in various matrices to further understand the pathophysiology of PML and define markers of survival. Four patients were included, of which three died in the few months following PML onset. Cerebrospinal fluid (CSF) viral loads were the highest, with plasma samples having lower viral loads and urine samples being mostly negative. Whether at PML onset or during follow-up, neutralizing antibody (NAb) titers directed against the same autologous strain (genotype or mutant) were the highest in plasma, with CSF titers being on average 430-fold lower and urine titers 500-fold lower at the same timepoint. Plasma NAb titers against autologous genotype or mutant were lower in non-survivor patients, though no neutralization “blind spot” was observed. The surviving patient was followed up until nine months after PML onset and presented, at that time, an increase in neutralizing titers, from 38-fold against the autologous genotype to around 200-fold against PML mutants. Our results suggest that patients’ humoral neutralizing response against their autologous strain may play a role in PML outcome, with survivors developing high NAb titers in both plasma and CSF.

Improved diagnosis of viral encephalitis in adult and pediatric hematological patients using viral metagenomics.

Metagenomic sequencing is a powerful technique that enables detection of the full spectrum of pathogens present in any specimen in a single test. Hence, metagenomics is increasingly being applied for detection of viruses in clinical cases with suspected infections of unknown etiology and a large number of relevant potential causes. This is typically the case in patients presenting with encephalitis, in particular when immunity is impaired by underlying disorders. In this study, viral metagenomics has been applied to a cohort of hematological patients with encephalitis of unknown origin. Because viral loads in cerebrospinal fluid of patients with encephalitis are generally low, the technical performance of a metagenomic sequencing protocol with viral enrichment by capture probes targeting all known vertebrate viral sequences was studied. Subsequently, the optimized viral metagenomics protocol was applied to a cohort of hematological patients with encephalitis of unknown origin. Viral enrichment by capture probes increased the viral sequence read count of metagenomics on cerebrospinal fluid samples 100 - 10.000 fold, compared to unenriched metagenomic sequencing. In five out of 41 (12%) hematological patients with encephalitis, a virus was detected by viral metagenomics which had not been detected by current routine diagnostics. BK polyomavirus, hepatitis E virus, human herpes virus-6 and Epstein Barr virus were identified by this unbiased metagenomic approach. This study demonstrated that hematological patients with encephalitis of unknown origin may benefit from early viral metagenomics testing as a single step approach.

Human immunodeficiency virus-associated vacuolar encephalomyelopathy with granulomatous-lymphocytic interstitial lung disease improved after antiretroviral therapy: a case report

BackgroundVacuolar encephalomyelopathy, a disregarded diagnosis lately, was a major neurological disease in the terminal stages of human immunodeficiency virus (HIV)-1 infection in the pre-antiretroviral therapy (ART) era. Granulomatous-lymphocytic interstitial lung disease (GLILD) was classically identified as a non-infectious complication of common variable immunodeficiency; however, it is now being recognized in other immunodeficiency disorders. Here, we report the first case of GLILD accompanied by vacuolar encephalomyelopathy in a newly diagnosed HIV-infected man.Case presentationA 40-year-old Japanese man presented with chronic dry cough and progressing paraplegia. Radiological examination revealed diffuse pulmonary abnormalities in bilateral lungs, focal demyelinating lesions of the spinal cord, and white matter lesions in the brain. He was diagnosed with GLILD based on marked lymphocytosis detecting in bronchoalveolar lavage, and transbronchial-biopsy proven T-cellular interstitial lung disease with granulomas. Microbiological examinations did not reveal an etiologic agent. The patient was also diagnosed with HIV-associated vacuolar encephalomyelopathy on the basis of an elevated HIV viral load in cerebrospinal fluid. After initiating ART, the brain lesions and paraplegia improved significantly, and interstitial abnormalities of the lungs and cough disappeared.ConclusionThis report highlights that even in the post-ART era in developed countries with advanced healthcare services, HIV-associated vacuolar encephalomyelopathy should be considered in the differential diagnosis of a progressive neurological disorder during the first visit. Furthermore, GLILD may represent an HIV-associated pulmonary manifestation that can be treated by ART.

A longitudinal study of brain volume changes in rhesus macaque model infected with SIV.

Given the current lack of understanding of brain volume changes caused by HIV infection, this study aimed to longitudinally assess the changes in regional brain tissue volume following HIV infection and to explore its relationship with peripheral blood absolute CD4+ lymphocyte count (CD4+), the percentage of monocytes in plasma(MON%) and cerebrospinal fluid viral load (CFVL).Four adult male rhesus monkeys were examined in healthy status and following infection with simian immunodeficiency virus using high-resolution 3D T1-weighted sagittal whole brain magnetic resonance imaging. DPABI and SPM were used to process and record changes in brain tissue volume. Correlation analyses were then used to explore the above relationships. Compared with brain tissue volume during the healthy stage, there was no change at 12 and 24weeks postinoculation (12wpi, 24wpi). At 36wpi, 48wpi, and 60wpi, basal ganglia, left inferior temporal gyrus, left occipital gyrus, and left superior frontal gyrus exhibited varying degrees of atrophy. There was no association found between CD4+, MON%, CFVL, and brain volume loss in any brain region. Our research demonstrated that in the early stage of HIV infection, local brain tissue atrophy can be demonstrated by MRI technique; furthermore, MRI can identify the earliest site of atrophy as well as the most severely affected site. Although there was no significant correlation between brain tissue volume loss and CD4+, MON%, and CFVL, our findings provided some evidence in the application of volumetric MR imaging in the early diagnosis and treatment follow-up of patients with HIV infection.

Two treatment strategies for management of Neurosymptomatic cerebrospinal fluid HIV escape in Pune, India.

Symptomatic cerebrospinal fluid (CSF) viral escape (sCVE) is reported in people with HIV, who are on ritonavir-boosted protease inhibitor (PI/r) containing antiretroviral therapy (ART). Management of sCVE includes performing genotypic HIV-1 resistance testing (GRT) on CSF and plasma HIV and changing ART accordingly. Neither GRT nor newer drugs (Dolutegravir and Darunavir/ritonavir) are routinely available in India. As a result, management of sCVE includes 2 modalities: a) ART intensification by adding drugs that reach therapeutic concentrations in CSF, like Zidovudine, to existing ART or b) Changing to a regimen containing newer boosted PI/r and integrase strand transfer inhibitor (INSTI) as per GRT or expert opinion. In this retrospective study, we report the outcomes of above 2 modalities in treatment of sCVE in Pune, India.Fifty-seven episodes of sCVE in 54 people with HIV taking PI/r-containing ART were identified. Clinical, demographic, laboratory and ART data were recorded. Forty-seven cases had follow-up data available after ART change including measurement of plasma and CSF viral load (VL).Of the 47 cases, 23 received zidovudine intensification (Group A, median VL: plasma- 290, CSF- 5200 copies/mL) and 24 received PI/INSTI intensification (Group B, median VL: plasma- 265, CSF-4750 copies/mL). CSF GRT was performed in 16 participants: 8 had triple class resistance. After ART change, complete resolution of neurologic symptoms occurred in most participants (Group A: 18, Group B: 17). In Group A, follow-up plasma and CSF VL were available for 21 participants, most of whom achieved virologic suppression (VL < 20 copies/mL) in plasma (17) and CSF (15). Four participants were shifted to the PI/INSTI intensification group due to virologic failure (plasma or CSF VL > 200 copies/mL). In Group B, follow-up plasma and CSF VL were available for 23 participants, most of whom also achieved virologic suppression in plasma (21) and CSF (18). Four deaths were noted, 2 of which were in individuals who interrupted ART.This is a unique sCVE cohort that was managed with 1 of 2 approaches based on treatment history and the availability of GRT. At least 75% of participants responded to either approach with virologic suppression and improvement in symptoms.

Simian Immunodeficiency Virus-Infected Memory CD4+ T Cells Infiltrate to the Site of Infected Macrophages in the Neuroparenchyma of a Chronic Macaque Model of Neurological Complications of AIDS.

Simian immunodeficiency virus (SIV)-infected nonhuman primates can serve as a relevant model for AIDS neuropathogenesis. Current SIV-induced encephalitis (SIVE)/neurological complications of AIDS (neuroAIDS) models are generally associated with rapid progression to neuroAIDS, which does not reflect the tempo of neuroAIDS progression in humans. Recently, we isolated a neuropathogenic clone, SIVsm804E-CL757 (CL757), obtained from an SIV-infected rhesus macaque (RM). CL757 causes a more protracted progression to disease, inducing SIVE in 50% of inoculated animals, with high cerebral spinal fluid viral loads, multinucleated giant cells (MNGCs), and perivascular lymphocytic cuffing in the central nervous system (CNS). This latter finding is reminiscent of human immunodeficiency virus (HIV) encephalitis in humans but not generally observed in rapid progressor animals with neuroAIDS. Here, we studied which subsets of cells within the CNS were targeted by CL757 in animals with neurological symptoms of SIVE. Immunohistochemistry of brain sections demonstrated infiltration of CD4+ T cells (CD4) and macrophages (MΦs) to the site of MNGCs. Moreover, an increase in mononuclear cells isolated from the brain tissues of RMs with SIVE correlated with increased cerebrospinal fluid (CSF) viral load. Subset analysis showed a specific increase in brain CD4+ memory T cells (Br-mCD4), brain-MΦs (Br-MΦs), and brain B cells (Br-B cells). Both Br-mCD4s and Br-MΦs harbored replication-competent viral DNA, as demonstrated by virus isolation by coculture. However, only in animals exhibiting SIVE/neuroAIDS was virus isolated from Br-MΦs. These findings support the use of CL757 to study the pathogenesis of AIDS viruses in the central nervous system and indicate a previously unanticipated role of CD4s cells as a potential reservoir in the brain.IMPORTANCE While the use of combination antiretroviral therapy effectively suppresses systemic viral replication in the body, neurocognitive disorders as a result of HIV infection of the central nervous system (CNS) remain a clinical problem. Therefore, the use of nonhuman primate models is necessary to study mechanisms of neuropathogenesis. The neurotropic, molecular clone SIVsm804E-CL757 (CL757) results in neuroAIDS in 50% of infected rhesus macaques approximately 1 year postinfection. Using CL757-infected macaques, we investigate disease progression by examining subsets of cells within the CNS that were targeted by CL757 and could potentially serve as viral reservoirs. By isolating mononuclear cells from the brains of SIV-infected rhesus macaques with and without encephalitis, we show that immune cells invade the neuroparenchyma and increase in number in the CNS in animals with SIV-induced encephalitis (SIVE). Of these cells, both brain macrophages and brain memory CD4+ T cells harbor replication-competent SIV DNA; however, only brain CD4+ T cells harbored SIV DNA in animals without SIVE. These findings support use of CL757 as an important model to investigate disease progression in the CNS and as a model to study virus reservoirs in the CNS.

What can characterization of cerebrospinal fluid escape populations teach us about viral reservoirs in the central nervous system?

To review the evidence that CSF (cerebrospinal fluid) escape populations are produced by viral reservoirs in the central nervous system (CNS). CSF escape is a rare phenomenon in which individuals on suppressive ART have well controlled systemic infections with elevated levels of HIV-1 RNA in their CSF. However, the rarity of CSF escape coupled with relatively low CSF viral loads has impeded detailed analyses of these populations. Here, and in a previous study, we performed genetic and phenotypic assessments of CSF escape populations to determine whether CSF escape is produced by CNS reservoirs or by cells trafficking through the CNS. We report HIV-1 viral loads in the CSF and blood plasma of four individuals with CSF escape (one new example and three previously described examples). We performed phylogenetic analyses of the viral env gene to evaluate diversity within the CSF escape populations and performed entry analyses to determine whether Env proteins were adapted to entering macrophage/microglia. Two individuals had CSF escape produced by CNS reservoirs. In contrast, the remaining two cases were likely because of transient viral production from cells migrating into the CNS and releasing virus. Together our analyses indicate that replication-competent HIV-1 can persist in the CNS during ART, but that not all cases of CSF escape are produced by CNS reservoirs. Our results also suggest that both CD4 T cells and macrophage/microglia can serve as persistent viral reservoirs in the CNS.

Neurocognitive Impairment in the Combined Antiretroviral Therapy Era in a Romanian Cohort of Young Adults with Chronic HIV Infection.

HIV-associated neurocognitive disorders (HAND) continue to be reported even in patients with successful antiretroviral treatment. We investigated the prevalence of neurocognitive impairment and possible HIV-associated determinants of cognition in a Romanian cohort of young adults, parenterally infected with HIV during their first years of life. Two hundred fourteen treatment-experienced HIV-positive individuals [median age: 24 years, males: 48%, median duration on combined antiretroviral therapy (cART): 12 years] underwent standard immunologic and virological monitoring and antiretroviral resistance testing using pol gene sequencing in both plasma and, when available, cerebrospinal fluid (CSF) paired samples. Neurocognitive impairment was assessed using a comprehensive neuropsychological test battery, and a global deficit score (GDS) was calculated (cutoff ≥0.5). Cognitive impairment was detected in 35% of the study participants, without any association with sex, median age, CD4 cell count (actual or nadir), CSF and plasma viral load (actual or zenith), AIDS diagnosis, duration of HIV infection, and cART characteristics. Participants carrying resistant viruses tended to be more frequently cognitively impaired (p = 0.36), with a higher median GDS value (p = 0.06) compared with participants harboring wild-type HIV, although the figures did not reach statistical significance. No signs of virological compartmentalization were observed based on CSF versus plasma viral load and on the profile of pol sequences. A moderate rate of mild neurocognitive impairment is still present in young adults with chronic HIV infection acquired in early childhood despite successful cART, without any association with classic markers of HIV infection. New biomarkers reflecting persistent central nervous system inflammation and neuronal injury may be more relevant for the development of HAND.

Practical approach to method verification in plasma and validation in cerebrospinal fluid under accreditation using a flexible scope in molecular virology: setting up the HIV, HBV and HCV Aptima™ Quant Dx assays.

Background Our laboratory obtained the ISO 15189 accreditation for the plasmatic HIV-1, HBV and HCV viral load (VL) using the m2000 RealTime™ system, which was recently changed for the platform Panther®. Here, we discuss a strategy for performing method validation/verification very quickly. Methods We performed the mandatory (repeatability, internal quality assessment [IQA], measurement uncertainty [MU]) and optional technical verifications for CE/IVD assays using the flexible scope range A. We also performed the mandatory assays for the validation of HIV-1 VL in the cerebrospinal fluid (CSF) using the flexible scope range B. The change was checked by following up on the turnaround time (TAT). Results The coefficient of variation (CV%) for repeatability and IQA complied with the limit of 0.25 log. The MU results ranged from 0.04 to 0.25 log copies or IU/mL. The comparisons of methods showed excellent correlations (R2 = 0.96 for the three parameters) but a delayed centrifugation on HCV VL showed variations of up to 2 log IU/mL. An excellent linearity for HIV-1 in the CSF was obtained from 1.5 to 5 log copies/mL with R2 = 0.99. The TAT increased (84%-98%) in routine usage. Conclusions The three Aptima assays are well suited for routine laboratory use and can be integrated within less than 2 weeks in accordance with flexible scope range A. Our data allows us to confidently perform HIV-1 VL in CSF following flexible scope range B. Finally, we provide an organizational guide for flexible scope management in molecular virology within a short time frame.

Neuroinflammatory Changes in Relation to Cerebrospinal Fluid Viral Load in Simian Immunodeficiency Virus Encephalitis.

The exact cause of neurocognitive dysfunction in HIV-positive patients despite successful control of the infection in the periphery is not completely understood. One suggested mechanism is a vicious cycle of microglial activation and release of proinflammatory chemokines/cytokines that eventually leads to neuronal loss and dysfunction. However, the exact role of microglial activation in the earliest stages of the infection with high cerebrospinal fluid (CSF) viral loads (VL) is unclear. In this study, we imaged the translocator protein (TSPO), a mitochondrial membrane receptor known to be upregulated in activated microglia and macrophages, in rhesus macaques before and multiple times after inoculation with a neurotropic simian immunodeficiency virus (SIV) strain (SIVsm804E), using 18F-DPA714 positron emission tomography (PET). The whole-brain standardized uptake values of TSPO at equilibrium reflecting total binding (SUVT) and binding potentials (BPND) were calculated and correlated with CSF and serum markers of disease, and a corresponding postmortem immunostaining analysis was also performed. SUVT was found to be inversely correlated with both CSF VL and monocyte chemoattractant protein 1 (MCP-1) levels. In SIV-infected macaques with very high CSF VL at necropsy (>106 copies/ml), we found decreased TSPO binding by PET, and this was supported by immunostaining which showed glial and neuronal apoptosis rather than microglial activation. On the other hand, with only moderately elevated CSF VL (∼104 copies/ml), we found increased TSPO binding as well as focal and diffuse microglial activation on immunostaining. Our results in the SIV-infected macaque model provide insights into the relationship between HIV neuropathology and CSF VL at various stages of the disease.IMPORTANCE Neurological and cognitive problems are a common complication of HIV infection and are prevalent even in treated individuals. Although the molecular processes underlying brain involvement with HIV are not completely understood, inflammation is suspected to play a significant role. Our work presents an in vivo assessment of neuroinflammation in an animal model of HIV, the simian immunodeficiency virus (SIV)-infected rhesus macaque. Using positron emission tomography (PET) imaging, we identified changes in brain inflammation after inoculation with SIV over time. Interestingly, we found decreased binding of the PET ligand in the presence of very high cerebrospinal fluid (CSF) viral loads. These findings were supported by immunostaining which showed marked glial loss instead of inflammation. This study provides insight into glial and neuronal changes associated with very high CSF viral load and could reflect similar changes occurring in HIV-infected patients.