RNA In Cerebrospinal Fluid Research Articles

Small rnas in cerebrospinal fluid samples from patients with Alzheimer's disease and neurologically normal controls

Aside from microRNA, very little is known about other small RNA species that can be detected in cerebrospinal fluid. For example, piwi-interacting RNAs are a highly diverse class of small RNA. They were recently identified to have expression outside of germline cells, and were found to be expressed in the brain. Changes in these other small RNAs have not yet been extensively studied with respect to the pathology associated with Alzheimer's disease. Cell-free RNA was isolated from 1 mL cerebrospinal fluid samples. As proof-of-principal, we sequenced the small RNA content from cerebrospinal fluid samples from patients with Alzheimer's disease, Parkinson's disease and neurologically normal control subjects (>50 subjects in each category). All subjects were postmortem and had full pathology reports. We assessed the small RNA findings with regards to pathology as well as confirmed disease diagnosis. We were able to detect several abundant small RNA species in addition to microRNAs that are consistently expressed in our cerebrospinal fluid samples, including small nucleolar RNAs, small nuclear RNAs and piwi-interacting RNAs. Using piwi-interacting RNAs as an example, we detected 7922 piRNAs across all cerebrospinal fluid samples. A total of 109 piwi-interacting RNAs were consistently detected in more than 90% of the samples, with a mean normalized expression of 549 and maximum of 15460 counts. When we examined piwi-interacting RNA levels between Alzheimer's patients and neurologically normal controls, we found several to be differentially expressed. We also found several differentially expressed piwi-interacting RNA between Alzheimer's and Parkinson's patients. These data demonstrate that, in addition to microRNA, there are other small RNA species that can be detected in cerebrospinal fluid. We identified piwi-interacting RNAs that were differentially expressed in cerebrospinal fluid samples from Alzheimer's disease patients when compared to controls and Parkinson's patients. In comparison with serum samples from the same individuals, there are nearly twice as many detected piwi-interacting RNA in cerebrospinal fluid. These data suggest that there may be additional small RNAs regulating the pathogenesis of Alzheimer's disease and that they are detectable in cerebrospinal fluid.

Detection of feline coronavirus in cerebrospinal fluid for diagnosis of feline infectious peritonitis in cats with and without neurological signs.

The objective of this study was to evaluate the sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) detecting feline coronavirus (FCoV) RNA in cerebrospinal fluid (CSF) of cats with and without neurological and/or ocular signs for the diagnosis of feline infectious peritonitis (FIP). This prospective case-control study included 34 cats. Nineteen cats had a definitive histopathological diagnosis of FIP (seven of these with neurological and/or ocular signs), and 15 cats had other diseases but similar clinical signs (three of these with neurological and/or ocular signs). Real-time RT-PCR was performed on the CSF of all cats, and sensitivity, specificity, and positive (PPV) and negative predictive values (NPV) were calculated. Real-time RT-PCR of CSF showed a specificity of 100% in diagnosing FIP, a sensitivity of 42.1%, a PPV of 100% and an NPV of 57.7%. The sensitivity of the real-time RT-PCR of CSF in cats with neurological and/or ocular signs was 85.7%. Although it is known that RT-PCR can give false positive results, especially if performed using serum or plasma, this real-time RT-PCR detecting FCoV RNA in CSF can be considered a reliable specific tool for the diagnosis of FIP. If only cats with neurological involvement are evaluated, the sensitivity of this real-time RT-PCR in CSF is also high.

Viral and inflammatory markers in cerebrospinal fluid of patients with HIV-1-associated neurocognitive impairment during antiretroviral treatment switch.

The aim of the study was to evaluate HIV-1 viral load (VL) and inflammatory markers in cerebrospinal fluid (CSF) and neurocognitive performance in patients with neurocognitive impairment (NCI) while they were receiving tenofovir (TDF)/ emtricitabine (FTC)/efavirenz (EFV) and after switching to a regimen with enhanced central nervous system (CNS) penetrability. This was a prospective, single-arm pilot study. HIV-1-infected patients with plasma viral suppression and HIV-associated NCI on a regimen including TDF/FTC/EFV were switched to abacavir (ABC)/lamivudine (3TC)/maraviroc (MVC). The Global Deficit Score (GDS) was used to score cognitive function at baseline and 48 weeks after treatment switch. Both CSF and blood samples were taken at baseline and between weeks 24 and 36 after switching. HIV-1 RNA in plasma and CSF was determined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Inflammatory biomarkers in CSF were measured by enzyme-linked immunosorbent assay (ELISA). A total of 71 patients receiving TDF/FTC/EFV were screened. Twelve of them (17%) had documented NCI, lacked the human leucocyte antigen (HLA)-B*57:01 haplotype and harboured Chemokine Receptor Type-5 (CCR5)-tropic virus. Eight patients had detectable HIV-1 RNA (between 2.7 and 41.6 HIV-1 RNA copies/mL) in CSF at baseline. All participants had elevated levels of neopterin and Monocyte Chemoattractant Protein 1 (MCP-1) in CSF at baseline. Eight out of 12 patients completed their follow-up assessment after treatment switch. The GDS decreased from 0.55 to 0.4 (P = 0.085). Median HIV-1 RNA in CSF decreased from 3.49 to 2.20 (P = 0.23). Among the inflammation markers in CSF, tumour necrosis factor (TNF)-α decreased significantly from median 0.51 to 0.35 pg/mL (P = 0.027), showing a correlation with the changes in neopterin, interferon (IFN)-γ and interleukin (IL)-6. Most patients with NCI receiving TDF/FTC/EFV had low-level viraemia and/or increased inflammatory markers in CSF. Treatment switching to an MVC-containing regimen with better CNS penetration resulted in a trend towards improvement in neurocognitive status and reduced TNF-α concentrations in CSF.

Lack of usutu virus RNA in cerebrospinal fluid of patients with encephalitis of unknown etiology, Tuscany, Italy.

Usutu virus (USUV) is an African mosquito-borne flavivirus associated with human neurological disorders in Europe. Recently, USUV introduction in Europe has been traced back to Eurasian blackbirds deaths in the Tuscany region of Italy in 1996. Ninety-six cerebrospinal fluid (CSF) samples from patients with encephalitis of unknown etiology diagnosed in 2010-2013 were screened to determine whether USUV circulates in humans in Tuscany. Using real-time polymerase chain reaction, no positive patient was found. USUV does not seem to cause neuroinvasive disorders in humans in Tuscany.

Allogeneic Transplantation with CCR5 Δ32/Δ32 Cord Blood Hematopoietic Cells in a HIV-Infected Patient

Allogeneic HCT from a CCR5 Δ32/Δ32 unrelated donor by Hutter et al provides the only evidence to date of long term control of HIV infection, and a compelling argument in favor of homozygous CCR5 Δ32reconstitution as the key mechanism driving resistance to infection 6 years after stopping antiretroviral therapy (ART). Low prevalence of Δ32/Δ32 genotype (<1%) and stringent HLA criteria for conventional donors made the search for Òpatient number 2Ó unsuccessful for years. Here, we describe a case of allogeneic HCT using CCR5 Δ32/Δ32 cord blood (CB) cells in a patient with diffuse large B-cell lymphoma (DLBCL) and HIV infection.A 36 year-old man with HIV-1 infection from 2009 was diagnosed with DLBCL stage IIA in 2012. The patient had no illnesses associated with AIDS, good disease control and adherence to ART, with <50 HIV-1 RNA copies/mL and 360 CD4 T-cells/μL at DLBCL onset. Prior to HCT, the patient received CHOP-R (x6), ESHAP-R (x3), an autologous transplant with BEAM conditioning, GEMOX (x3), and abdominal radiotherapy. At HCT the patient had a refractory lesion in the left psoas, no B symptoms and good clinical performance. Baseline HIV-1 latent reservoir tests showed DNA copies in bulk and resting CD4 T cells and in GALT, CD4 T cell-associated HIV-1 RNA, free HIV-1 RNA in cerebrospinal fluid, replication competent viral size of 1.2 copies/107 CD4 T cells, and SCA (single copy assay) of 303 copies/mL. Patient’s HIV-1 was CCR5 tropic by both genotypic and phenotypic analyses.No HLA-matched sibling donors were available. Two CCR5 Δ32/Δ32 CB units (CBU) identified at the StemCyte inventory (Covina, CA) were HLA-compatible with the patient at the 4/6 level. A myeloablative CB alloHCT was performed using the single CCR5 Δ32/Δ32 CBU with higher cellularity (#1; Table 1) plus CD34+ cells from a haploidentical brother. The patient provided informed consent and IRB approved the alloHCT and research protocol. During HCT, ART combined abacavir, lamivudine and raltegravir. Neutrophil engraftment, 100% derived from the haploidentical donor, occurred on day +11. Post-thawing analysis of CBU#1 showed very poor clonogenic capacity, and donor switch to CB-origin failed, with persistently low CB-chimera <5% for up to 7 weeks (Figure 1). CBU#2 was infused on day +52, leading to a very rapid increase in CBU#1 chimerism, to 20% by day +59, 83% by day +66 and 100% by day +73. At this point, the patient’s hematopoiesis, including CD4 T-lymphocytes, became CCR5 Δ32/Δ32. Ultrasensitive plasma viral load analysis decreased right after HCT, reaching minimal levels at the time of full CB-chimera. No HIV-1 DNA was detected using droplet digital PCR quantification or semiquantitative tests of amplification. In addition, although recipient CD4 T cells responded to proliferation and activation stimuli, they were resistant to infection by the patient’s viral isolate as well as laboratory-adapted HIV-1 strains. Regretfully, our patient developed an aggressive DLBCL progression, and following very rapid clinical deterioration died from disease progression three months after HCT.Long term monitoring and target organs and tissues analysis of viral reservoir will not be possible in this case. However, our data suggest that CCR5 Δ32/Δ32 CB HCT can successfully eliminate HIV-1 and render the recipient’s T cells resistant to HIV-infection. Thus, they strongly support the use of CB as a platform for a broader application of this curative technology to other HIV-1 infected individuals.Abstract 1261. Table 1Patient, Donor and Cell Product CharacteristicsRecipientHaploidentical BrotherCBU #1CBU #2Year of birth or collection1975197120082008CCR5 genotypeWT / WTWT / Δ32Δ32 / Δ32Δ32 / Δ32HLA typing- HLA A01:01 / 68:0111 / 6801:01 / 03:0101:01 / 02:XX- HLA B08:01 / 07:0235 / 0708:01 / 07:0208:01 / 07:02- HLA DR03:01 / 13:0101 / 1303:01 / 14:0103:01 / 14:01Cell products contentPre-HCT ControlsPost Thawing #Pre-HCT ControlsPost Thawing #CD34 (x105/kg)−40 ¤0.70.520.730.22TNC (x107/kg)−−2.532.051.981.64CFU-GM (x104/kg)−−1.02 *0.110.12 *NilTotal CFU (x104/kg)−−2.13 *0.210.42 *Nil¤ Purified by Miltenyi positive selection to include <1 x104/kg CD3+ cells; * Quality controls performed prior to HCT at the transplant center’s laboratory in aliquot samples from segments attached to the units, as data were not available in the reports from the cord blood bank; # Subsequent quality controls in the final cell product after thawing the CBU for infusion. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Analysis of neurocognitive function and CNS endpoints in the PROTEA trial: darunavir/ritonavir with or without nucleoside analogues.

IntroductionDuring treatment with protease inhibitor monotherapy, the number of antiretrovirals with therapeutic concentrations in the cerebrospinal fluid (CSF) is lower, compared to standard triple therapy. However, the clinical consequences are unclear.MethodsA total of 273 patients with HIV RNA <50 copies/mL for over 24 weeks on current antiretrovirals randomized to darunavir/ritonavir (DRV/r) 800/100 mg once-daily, either as monotherapy (n=137) or with 2NRTIs (n=136). Neurocognitive function was evaluated in all patients by the Hopkins Verbal Learning Tests, the Colour Trail Tests and the Grooved Pegboard Test at screening, baseline and at Week 48. A global neurocognitive score (NPZ-5) was derived by averaging the standardized results of the five domains. In a central nervous system (CNS) sub-study (n=70), HIV RNA levels in the CNS were evaluated at baseline and Week 48. Clinical adverse events related to the CNS were collected at each visit.ResultsPatients were 83% male and 88% White, with median age 43 years. There were more patients with nadir CD4 count below 200 cells/µL in the DRV/r monotherapy arm (41/137, 30%) than the triple therapy arm (30/136, 22%). At Week 48, there was no difference between the treatment arms for the five combined domains of the neurocognitive score. At Week 48, the percentage of patients with an abnormal neurocognitive score among the five domains was 12.2% for DRV/r monotherapy and 14.9% for triple therapy. However, one patient on DRV/r monotherapy with a CD4 nadir of 17 cells/µL was hospitalized with HIV encephalomyelitis at Week 24, with HIV RNA 2500 copies/mL in the CSF and 125 copies/mL in the plasma. Symptoms resolved after intensification with high dose zidovudine. A second patient on DRV/r monotherapy with CD4 nadir of 166 cells/µL had a rise in HIV RNA in CSF from <40 copies/mL at baseline to 654 copies/mL at Week 48, with concurrent plasma HIV RNA of 77 copies/mL.ConclusionsIn this study for patients with HIV RNA <50 copies/mL at baseline, there was no difference in neurocognitive function between the treatment arms. However two patients on PI monotherapy with CD4 nadir <200 cells/µL developed viraemia in both CSF and plasma, with one symptomatic case. DRV/r monotherapy should be used with caution in patients with nadir CD4 counts below 200 cells/µL.

Low levels of HIV-1 RNA detected in the cerebrospinal fluid after up to 10 years of suppressive therapy are associated with local immune activation.

Though combination antiretroviral therapy reduces the concentration of HIV-1 RNA in both plasma and cerebrospinal fluid (CSF) below the detection limit of clinical assays, low levels of HIV-1 RNA are frequently detectable in plasma using more sensitive assays. We examined the frequency and magnitude of persistent low-level HIV-1 RNA in CSF and its relation to the central nervous system (CNS) immune activation. CSF and plasma HIV-1 RNA were measured using the single-copy assay with a detection limit of 0.3 copies/ml in 70 CSF and 68 plasma samples from 45 treated HIV-1-infected patients with less than 40 copies/ml of HIV-1 RNA in both fluids by standard clinical assays. We also measured CSF neopterin to assess intrathecal immune activation. Theoretical drug exposure was estimated using the CNS penetration-efficacy score of treatment regimens. CSF HIV-1 RNA was detected in 12 of the 70 CSF samples (17%) taken after up to 10 years of suppressive therapy, compared to 39 of the 68 plasma samples (57%) with a median concentration of less than 0.3 copies/ml in CSF compared to 0.3 copies/ml in plasma (P < 0.0001). CSF samples with detectable HIV-1 RNA had higher CSF neopterin levels (mean 8.2 compared to 5.7 nmol/l; P = 0.0085). Patients with detectable HIV-1 RNA in CSF did not differ in pretreatment plasma HIV-1 RNA levels, nadir CD4 cell count or CNS penetration-efficacy score. Low-level CSF HIV-1 RNA and its association with elevated CSF neopterin highlight the potential for the CNS to serve as a viral reservoir and for persistent infection to cause subclinical CNS injury.

Distinct systemic and central nervous system disease patterns in enterovirus and parechovirus infected children

Enteroviruses (EV) and human parechoviruses (HPeV) infections are increasingly identified in neonates and young children with sepsis, meningitis and encephalitis. We investigated EV and HPeV viral loads in plasma and cerebrospinal fluid (CSF) among those presenting with sepsis or central nervous system (CNS) disease to gain understanding of the nature of these infections. Detections frequencies and viral loads of EV and HPeV RNA were compared in plasma and CSF obtained from infected children originally identified on sepsis or CNS screening. Two distinct infection profiles were identified; 11 subjects with CNS disease, showed higher or similar viral loads in CSF than in plasma (median plasma:CSF ratio 0.5), whereas 14 children with sepsis showed low or undetectable viral loads in CSF and high viral loads in plasma (mean ratio 5700). HPeV type 3 and one EV serotype (coxsackievirus B2) were primarily associated with the latter presentation. Simple detection of EV or HPeV RNA in CSF is not predictive of CNS disease, especially in the absence of clinical markers (i.e. pleocytosis). Screening of plasma can identify EV and HPeV RNA in a substantial proportion of sepsis cases, some of which will be missed if CSF samples alone are screened.

Laboratory and Clinical Characteristics of Human West Nile Virus Infections during 2011 Outbreak in Southern Greece

During the summer-autumn of 2011, a human outbreak of West Nile virus (WNV) infection occurred in southern Greece, following the first outbreak during 2010 in northern Greece. An investigation was performed to analyze laboratory diagnosis, geographic distribution, and clinical features of WNV cases in southern Greece. Serum and cerebrospinal fluid (CSF) specimens from all patients seeking laboratory diagnosis for suspected WNV infection were tested for the presence of specific WNV immunoglobulin M (IgM) and IgG antibodies. Detection of WNV RNA in CSF and whole blood samples was accomplished by real-time PCR. During August-October of 2011, 31 confirmed or probable cases of WNV infection were identified. In 25 of them, individuals experienced severe neurological manifestations and were classified as WNV neuroinvasive disease cases. Risk factors such as advanced age, hypertension, and diabetes mellitus were identified in most cases with neurological complications. As many as 25 of the WNV cases occurred in the broader region of Athens; the majority of them (17 cases) were identified in municipalities of Eastern Attica, located almost 40 km from the metropolitan area of Athens and 500 km from Central Macedonia, where the 2010 WNV outbreak occurred. The spread of the virus in a newly affected area of the country suggests that WNV has been established in Greece and disease transmission will be continued in the future.

Human parechovirus type 3 central nervous system infections in Israeli infants

IntroductionHuman parechoviruses (HPeV) have been recognized as the causative agents of central nervous system (CNS) infection of infants and young children in different parts of the world. The role of HPeV in CNS infection of Israeli infants and children is unknown. ObjectivesTo assess the detection rate of HPeV in enterovirus RT-PCR-negative cerebrospinal fluid (CSF) samples obtained during the years 2007–2009 from children 0–5 years old with suspected CNS infection or from very young infants with unexplained fever in four medical centers in Israel. Study designA total of 367 CSF samples were retrospectively tested for the presence of HPeV RNA using nested RT-PCR assay. Positive samples were further typed on the basis of molecular sequencing. Retrospective analysis of the medical charts was performed. ResultsHPeV3 RNA was detected in CSF obtained between May and September 2008 in 13 patients, all of whom were <3 months old (3.5% of all CSFs; 11.3% of all infants <3 months in 2008). The HPeV-positive CSF samples were without pleocytosis. All HPeV3-positive patients recovered without obvious short term sequelae. ConclusionHPeV infection could play an important role in summertime febrile/CNS illness in young infants during specific years with high HPeV activity. PCR detection of parechoviral RNA in CSF should be included in the diagnostic evaluation of fever or CNS infection of neonates and very young infants. The rapid identification of HPeV in CSF could curtail unnecessary empirical antibiotic treatment and shorten hospital stay in selected patients.

Comment on “Higher CNS Penetration-Effectiveness of Long-Term Combination Antiretroviral Therapy is Associated With Better HIV-1 Viral Suppression in Cerebrospinal Fluid”

Objectives: To determine HIV-1 RNA in cerebrospinal fluid (CSF) of successfully treated patients and to evaluate if combination antiretroviral treatments with higher central nervous system penetration-effectiveness (CPE) achieve better CSF viral suppression. Methods: Viral loads (VLs) and drug concentrations of lopinavir, atazanavir, and efavirenz were measured in plasma and CSF. The CPE was calculated using 2 different methods. Results: The authors analyzed 87 CSF samples of 60 patients. In 4 CSF samples, HIV-1 RNA was detectable with 43-82 copies per milliliter. Median CPE in patients with detectable CSF VL was significantly lower compared with individuals with undetectable VL: CPE of 1.0 (range, 1.0-1.5) versus 2.3 (range, 1.0-3.5) using the method of 2008 (P = 0.011) and CPE of 6 (range, 6-8) versus 8 (range, 5-12) using the method of 2010 (P = 0.022). The extrapolated CSF trough levels for atazanavir (n = 12) were clearly above the 50% inhibitory concentration (IC50) in only 25% of samples; both patients on atazanavir/ritonavir with detectable CSF HIV-1 RNA had trough levels in the range of the presumed IC50. The extrapolated CSF trough level for lopinavir (n = 42) and efavirenz (n = 18) were above the IC50 in 98% and 78%, respectively, of samples, including the patients with detectable CSF HIV-1 RNA. Conclusions: This study suggests that treatment regimens with high intracerebral efficacy reflected by a high CPE score are essential to achieve CSF HIV-1 RNA suppression. The CPE score including all drug components was a better predictor for treatment failure in the CSF than the sole concentrations of protease inhibitor or non-nucleoside reverse transcriptase inhibitor in plasma or CSF.

Higher CNS Penetration-Effectiveness of Long-term Combination Antiretroviral Therapy Is Associated With Better HIV-1 Viral Suppression in Cerebrospinal Fluid

To determine HIV-1 RNA in cerebrospinal fluid (CSF) of successfully treated patients and to evaluate if combination antiretroviral treatments with higher central nervous system penetration-effectiveness (CPE) achieve better CSF viral suppression. Viral loads (VLs) and drug concentrations of lopinavir, atazanavir, and efavirenz were measured in plasma and CSF. The CPE was calculated using 2 different methods. The authors analyzed 87 CSF samples of 60 patients. In 4 CSF samples, HIV-1 RNA was detectable with 43-82 copies per milliliter. Median CPE in patients with detectable CSF VL was significantly lower compared with individuals with undetectable VL: CPE of 1.0 (range, 1.0-1.5) versus 2.3 (range, 1.0-3.5) using the method of 2008 (P = 0.011) and CPE of 6 (range, 6-8) versus 8 (range, 5-12) using the method of 2010 (P = 0.022). The extrapolated CSF trough levels for atazanavir (n = 12) were clearly above the 50% inhibitory concentration (IC50) in only 25% of samples; both patients on atazanavir/ritonavir with detectable CSF HIV-1 RNA had trough levels in the range of the presumed IC50. The extrapolated CSF trough level for lopinavir (n = 42) and efavirenz (n = 18) were above the IC50 in 98% and 78%, respectively, of samples, including the patients with detectable CSF HIV-1 RNA. This study suggests that treatment regimens with high intracerebral efficacy reflected by a high CPE score are essential to achieve CSF HIV-1 RNA suppression. The CPE score including all drug components was a better predictor for treatment failure in the CSF than the sole concentrations of protease inhibitor or nonnucleoside reverse transcriptase inhibitor in plasma or CSF.

Cerebrospinal fluid HIV escape associated with progressive neurologic dysfunction in patients on antiretroviral therapy with well controlled plasma viral load

To characterize HIV-infected patients with neurosymptomatic cerebrospinal fluid (CSF) 'escape', defined as detectable CSF HIV RNA in the setting of treatment-suppressed plasma levels or CSF RNA more than 1-log higher than plasma RNA. Retrospective case series. Four urban medical centers in the United States and Europe. Virologically controlled HIV-infected patients on antiretroviral therapy (ART) with progressive neurologic abnormalities who were determined to have CSF 'escape'. INTERVENTION Optimization of ART based upon drug susceptibility and presumed central nervous system exposure. Levels of CSF HIV RNA and inflammatory markers, clinical signs and symptoms, and MRI findings. Ten patients presented with new neurologic abnormalities, which included sensory, motor, and cognitive manifestations. Median CSF HIV RNA was 3900 copies/ml (range 134-9056), whereas median plasma HIV RNA was 62 copies/ml (range <50 to 380). Median CD4 T-cell count was 482 cells/μl (range 290-660). All patients had been controlled to less than 500 copies/ml for median 27.5 months (range 2-96) and five of 10 had been suppressed to less than 50 copies/ml for median 19.5 months (range 2-96). Patients had documentation of a stable ART regimen for median 21 months (range 9-60). All had CSF pleocytosis or elevated CSF protein; seven of eight had abnormalities on MRI; and six of seven harbored CSF resistance mutations. Following optimization of ART, eight of nine patients improved clinically. The development of neurologic symptoms in patients on ART with low or undetectable plasma HIV levels may be an indication of CSF 'escape'. This study adds to a growing body of literature regarding this rare condition in well controlled HIV infection.

117. Insight into the role of CD8 T cells in HIV-associated neurocognitive impairment

Previous studies of HIV-associated neurocognitive disease (HAND) have focused on the role of monocytes/macrophages. Having observed that high CD8 levels are a risk factor for HAND and hypothesizing that cerebrospinal fluid (CSF) could be a portal of entry for activated CD8 T-cells, we investigated cytokine responses by CSF T-cells. HIV+ participants with and without HAND were treated with CNS-penetrating anti-retroviral agents. CSF cells and PBMCs were harvested before treatment and at 6 and 16 weeks post-treatment and assessed for intracellular expression of IL-2, IFNg and TNFa, in response to HIV antigens. Data were examined both longitudinally (16 subjects with 3 visits) and cross-sectionally (30 subjects, total 59 visits). Overall, CD4 and CD8 T-cells in the CSF were more likely to make all three cytokines than CD4 and CD8 T-cells in blood. Impairment was associated with higher levels of constitutive and HIV-induced IFNg (p = .04) and TNFa (p = .05) expression by CD8 T-cells in the CSF. While higher levels of HIV RNA in CSF suppressed CD8 cytokine expression in unimpaired subjects, impaired subjects persisted with high cytokine expression regardless of RNA levels (p = .04). In addition to identifying a novel mechanism of HIV pathogenesis, these findings validate viral suppression in both CSF and the periphery as an important treatment goal for HAND, but also suggest that modulation of the CD8 T-cell response may be required.

Influenza A 2009 H1N1 encephalitis in adults with viral RNA in cerebrospinal fluid

We present 3 cases of influenza A 2009 H1N1 encephalitis occurring in adults, corroborated by viral genome detection in cerebrospinal fluid (CSF) and evidence of meningeal inflammation on brain histopathology. This paper emphasizes the possibility of viral detection in CSF and stresses the lack of an effective treatment for neurological manifestations of influenza.

Factors associated with cerebrospinal fluid HIV RNA in HIV infected subjects undergoing lumbar puncture examination in a clinical setting

Cerebrospinal fluid (CSF) HIV RNA load may be associated with central nervous system (CNS) disease in HIV infected subjects. We investigated parameters associated with CSF HIV RNA within a large clinical cohort. All HIV infected subjects undergoing CSF examination including assessment of CSF HIV RNA at St. Mary's Hospital, London, UK between January 2008 and October 2010 were included. Parameters associated with a detectable CSF HIV RNA load were assessed using linear regression modelling. CSF viral escape was defined as CSF RNA >0.5 log(10) copies/mL greater than plasma HIV RNA and >200 copies/mL where plasma HIV RNA <50 copies/mL. Of 142 subjects, 99 were receiving antiretroviral therapy (ART). Plasma HIV RNA was <50 copies/mL in 69 subjects. CSF examination was performed for investigation of presumed HIV encephalopathy (IxHE, n = 57), other CNS diseases considered HIV related (n = 39), syphilis (n = 20) and CNS presentations not considered HIV related (n = 26). CSF viral escape was present in 30/142 (21%) subjects overall and in 9/69 (13%) of those on ART with undetectable plasma HIV RNA. Overall, plasma HIV RNA load was significantly associated with detectable CSF HIV RNA (p ≤ 0.001). In subjects with plasma HIV RNA <50 copies/mL, only CNS penetration effectiveness (CPE, 2008) score of <2 was significantly associated with detectable CSF HIV RNA (p = 0.044). In patients undergoing LP for IxHE both plasma HIV RNA and CPE scores were independently associated with detectable CSF HIV RNA (p = 0.019 & 0.003 respectively) which was not observed in subjects undergoing CSF examination for other medical reasons. In a clinical setting, CSF viral escape is observed frequently in 21% of subjects and is associated with different parameters depending on the clinical scenario.

Detection of enterovirus RNA in cerebrospinal fluid: Comparison of two molecular assays

Enterovirus (EV) and human parechovirus (HPeV) are a major cause of infection in childhood. A rapid diagnostic test may improve the management of patients with EV and HPeV infection. The aim of this study is to evaluate the performance of the GeneXpert enterovirus assay (GXEA) for detection of EV RNA compared to a user-developed reverse-transcriptase (RT) quantitative real-time PCR (qPCR) in routine clinical practice. Also a RT-qPCR assay for detection of HPeV RNA in different clinical samples was developed and evaluated. Cerebrospinal fluid (CSF) from 232 patients suspected for meningitis was collected and tested for EV and HPeV using RT-qPCR assays. In parallel an aliquot of the samples was tested using the GXEA and viral culture. EV RNA was detected in 22 (19.0%) and 28 (24.1%) of 116 samples using the GXEA and RT-qPCR assay, respectively. EV was isolated from 10 of 116 (8.6%) samples by viral culture. GXEA had a sensitivity, specificity, positive predictive value and negative predictive value of 82.1%, 100%, 100% and 96.2%, respectively. In this study, molecular assays were superior to viral culture for detecting EV RNA in CSF. GXEA showed a high specificity but a lower sensitivity for the detection of EV RNA compared to the RT-qPCR assay.

Comparative detection of enterovirus RNA in cerebrospinal fluid: GeneXpert system vs. real-time RT-PCR assay

Enteroviruses (EVs) constitute the most common cause of aseptic meningitis in both children and adults. Molecular techniques have now been recognized as the reference standard for the diagnosis of EV infections, and the rapidity of the molecular diagnosis of EV meningitis has been shown to be a determining factor in the management of patients. The rapid documentation of EV RNA in cerebrospinal fluid (CSF) is key to adapting patient management and the therapeutic regimen. To shorten the time needed for virological documentation, we implemented EV RNA detection in two point-of-care (POC) laboratories. Here, we present the results of the POC detection of EV RNA with the Xpert EV kit on the GeneXpert integrated system, and a comparison with the real-time RT-PCR (rtRT-PCR) assay routinely used in the core virology laboratory. From January to September 2009, a total of 310 CSF samples were tested. The rtRT-PCR gave 81 positive, 225 negative and four ‘indeterminate’ results. POC results were concordant in 81.6% (253/310). Most of the discrepancies consisted of ‘indeterminate’ results at the POC level (16%). Calculated performances (excluding the indeterminate results) of the Xpert EV kit on the GeneXpert system in POC settings were 100%, 98.9%, 97.6% and 100% for Sensibility, Specificity, positive predictive value and negative predictive value, respectively. Taken together, these results indicate that the implementation of POC detection of EV RNA can provide robust results in <4 h, and may have a significant impact on patient management, therapeutic attitude, and hospitalization costs.

Analysis of correlation between cerebrospinal fluid and plasma HIV-1 RNA levels in patients with neurological opportunistic diseases

The question of whether HIV-1 RNA in cerebrospinal fluid (CSF) is derived from viral replication in the central nervous system or simply reflects the transit of infected lymphocytes from the blood compartment has long been a matter of debate. Some studies found no correlation between CSF and plasma viral load, whereas others did. The lack of a correlation between the two compartments suggests that the presence of HIV-1 RNA is not simply due to the passive passage of the virus from blood to CSF but rather due to intrathecal replication. To evaluate the correlation between plasma and CSF HIV-1 RNA levels and to identify situations in which there is no correlation between the two compartments, seventy patients were prospectively studied. The association between CSF and plasma viral load was evaluated in the total population and in subgroups of patients with similar characteristics. A correlation between the CSF and plasma compartments was observed for patients undergoing highly active antiretroviral therapy (HAART), those with a CD4 T lymphocyte count lower than 200 cells/mm³, and those with increased CSF protein content. On the other hand, no correlation was observed for patients without adequate virological control, who had a CD4 count higher than 200 cells/mm³ and who did not use HAART. The correlation between the two compartments observed in some patients suggests that CSF HIV-1 RNA levels may reflect plasma levels in these subjects. In contrast, the lack of a correlation between the two compartments in patients who were not on HAART and who had normal CSF proteins and a poor virological control possibly indicates compartmentalization of the virus in CSF and, consequently, plasma-independent intrathecal viral replication.

Detection of mumps virus RNA in cerebrospinal fluid of patients with neuromyelitis optica

Neuromyelitis optica (NMO) is an acute inflammatory disease that preferentially involves the optic nerves and spinal cord. Although many infectious agents, including mumps virus, are postulated to have a role in the pathogenesis of multiple sclerosis (MS), the relationship between NMO and infectious agents remains uncertain. To investigate the relationship between NMO and viruses that have special affinity for the central nervous system, we performed a nested polymerase chain reaction (PCR) to detect mumps virus or enterovirus RNA in cerebrospinal fluid samples from 13 patients with MS, 8 with NMO and 20 with other neurological diseases (ONDs). Nested PCR was positive for mumps virus in 2 (25%) of NMO patients, but in none of those with MS and ONDs. Moreover, nested PCR results became negative in the remission phase in the two PCR-positive NMO patients. Mumps virus may have some role in the pathogenesis of NMO.