HIV Encephalitis Research Articles

HIV-1 Clade-Specific Differences in the Induction of Neuropathogenesis

Human immunodeficiency virus (HIV)-associated dementia (HAD) is common among clade B HIV-infected individuals, but less common and less severe among individuals infected with clade C HIV-1, suggesting clade-specific differences in neuropathogenicity. Although differences in neuropathogenicity have been investigated in vitro using viral proteins responsible for HAD, to date there are no virological studies using animal models to address this issue. Therefore, we investigated neuropathogenesis induced by HIV-1 clades using the severe combined immune deficiency (SCID) mouse HIV encephalitis model, which involves intracranial injection of macrophages infected with representative clade B (HIV-1(ADA)) or clade C (HIV-1(Indie-C1)) HIV-1 isolates into SCID mice. In cognitive tests, mice exposed to similar inputs of HIV-1 clade C made fewer memory errors than those exposed to HIV-1 clade B. Histopathological analysis of mice exposed to clade B exhibited greater astrogliosis and increased loss of neuronal network integrity. In vitro experiments revealed differences in a key characteristic of HIV-1 that influences HAD, increased monocyte infiltration. HIV-1(Indie-C1)-infected macrophages recruited monocytes poorly in vitro compared with HIV-1(ADA)-infected macrophages. Monocyte recruitment was HIV-1 Tat and CCL2 dependent. This is the first demonstration, ever since HIV neuropathogenesis was first recognized, that viral genetic differences between clades can affect disease severity and that such studies help identify key players in neuropathogenesis by HIV-1.

CCL5/RANTES Gene Deletion Attenuates Opioid-Induced Increases in Glial CCL2/MCP-1 Immunoreactivity and Activation in HIV-1 Tat-Exposed Mice

To assess the role of CC-chemokine ligand 5 (CCL5)/RANTES in opiate drug abuse and human immunodeficiency virus type 1 (HIV-1) comorbidity, the effects of systemic morphine and intrastriatal HIV-1 Tat on macrophage/microglial and astroglial activation were assessed in wild-type and CCL5 knockout mice. Mice were injected intrastriatally with vehicle or Tat and assessed after 7 days. Morphine was administered to some Tat-injected mice via time-release implant (5 mg/day, s.c. for 5 days) starting at 2 days post injection. Glial activation was significantly reduced in CCL5(-/-) compared to wild-type mice at 7 days following combined Tat and morphine exposure. Moreover, the percentage of 3-nitrotyrosine immunopositive macrophages/microglia was markedly reduced in CCL5(-/-) mice injected with Tat +/- morphine compared to wild-type counterparts, suggesting that CCL5 contributes to nitrosative stress in HIV-1 encephalitis. In CCL5(-/-) mice, the reductions in Tat +/- morphine-induced gliosis coincided with significant declines in the proportion of CCL2/MCP-1-immunoreactive astrocytes and macrophages/microglia compared to wild-type counterparts. In knockout mice, neither Tat alone nor in combination with morphine increased the proportion of CCL2-immunoreactive astrocytes above percentages seen in vehicle-injected controls. Macrophages/microglia differed showing modest, albeit significant, increases in the proportion of CCL2-positive cells with combined Tat and morphine exposure, suggesting that CCL5 preferentially affects CCL2 expression by astroglia. Thus, CCL5 mediates glial activation caused by Tat and morphine, thereby aggravating HIV-1 neuropathogenesis in opiate abusers and non-abusers. CCL5 is implicated as mediating the cytokine-driven amplification of CCL2 production by astrocytes and resultant macrophage/microglial recruitment and activation.

Cell specific temporal infection of the central nervous system in a simian immunodeficiency virus model of human immunodeficiency virus encephalitis

Increasing evidence supports early central nervous system (CNS) infection by human immunodeficiency virus (HIV) and subsequent establishment of a brain viral reservoir. Definitive temporal studies determining when and within which CNS cells viral DNA is present are lacking. This study utilized simian immunodeficiency virus (SIV) infected macaques sacrificed at days 10, 21, 56 and 84 post-inoculation. Laser microdissection isolated pure perivascular macrophage, parenchymal microglia and astrocyte populations. Nested PCR and sequencing determined the presence and characteristics of SIV env V3 and V1 DNA from each population.

SIV/HIV latency in the CNS: role of innate immune responses

In order to model the molecular events that lead to HIV infection and pathogenesis in the brain, we developed an accelerated, consistent simian model of HIV infection in which pigtailed macaques are inoculated with a neurovirulent SIV clone and an immunosuppressive SIV strain. Infected animals invariably develop AIDS and over 90% develop central nervous system disease with lesions typical of HIV encephalitis as well as peripheral nervous system disease with neurodegeneration by 3 months postinoculation. This model has allowed us to identify early events in the brain that lead to the downregulation of SIV replication and the establishment of latency in macrophages. We have demonstrated that SIV infection is established in the brain during acute infection and this is accompanied by increased levels of inflammatory cytokines/chemokines and increased expression of macrophages inflammatory markers. Further, our studies have delineated the effects of IFNβ in suppressing early virus replication, suppressing virus transcription and inducing viral latency as well as downregulation of early inflammation. We have identified a novel pathway of IFNβ signaling that affects viral gene expression as well as expression of cellular genes involved in inflammation. The molecular mechanisms involved in these processes will be discussed.

OTK18 Levels in Plasma and Cerebrospinal Fluid Correlate with Viral Load and CD8 T-cells in Normal and AIDS Patients

OTK18 is a C2H2 type zinc finger protein expressed by human macrophages following HIV infection. OTK18 possesses antiretroviral activity, and its processing products accumulate in the cytoplasm of perivascular brain macrophages in advanced HIV encephalitis cases. Since the regulation of OTK18 expression in living patients following human immunodeficiency virus-1 (HIV-1) infection is unknown, our objective is to investigate the first cohort study on OTK18 protein levels in living patients. We assessed OTK18 levels in plasma and cerebrospinal fluid (CSF) in 44 living patients with or without HIV-1 infection, with diverse demographic and clinical background. A novel high-sensitivity OTK18 ELISA system was developed to measure OTK18 levels in CSF and plasma using custom made biotinylated monoclonal antibodies against OTK18. The correlation of OTK18 levels with epidemiological parameters was statistically analyzed. Multiple linear regression modeling suggested that plasma OTK18 levels for HIV-1-positive subjects were only about one sixth of that for HIV-1-negative subjects. Higher CD8 T-cell counts were associated with higher levels of OTK18. Using proportional odds logistic regression, we showed that HIV-1-positive patients have significantly lower OTK18 in CSF samples, but we did not observe significant correlation between CD8 T-cell counts and CSF OTK18 levels. OTK18 levels in both plasma and CSF are significantly lower in HIV-1-positive subjects as compared to HIV-1-negative subjects. Plasma OTK18 levels are positively correlated to CD8 T-cell counts, independent of HIV-1 status.

Osteopontin Is Increased in HIV‐Associated Dementia

Since the introduction of highly active antiretroviral therapy, survival rates for human immunodeficiency virus (HIV) infection have markedly improved, but less of an effect has been found for HIV-associated neurocognitive disorders. On the basis of our previous findings, we hypothesized that increased production of osteopontin might contribute to the persistence of central nervous system (CNS) dysfunctions. We found increased levels of osteopontin in the brains of humans with HIV encephalitis and monkeys with simian immunodeficiency virus (SIV) encephalitis. In cerebrospinal fluid, osteopontin levels were found to be elevated in HIV-infected individuals, regardless of their neuropsychological status. However, plasma osteopontin levels were significantly increased in individuals with HIV-associated dementia. In addition, a longitudinal study of monkeys revealed that plasma levels of osteopontin increased before the development of SIV-induced neurological and clinical abnormalities. Thus, plasma levels of osteopontin are significantly correlated with HIV-induced CNS dysfunction in the current era of efficacious antiviral treatment, and this finding suggests that the development of interventions to modulate osteopontin production or signaling might be beneficial in the prevention or treatment of HIV-induced CNS disorders.

Cannabinoids as therapeutic agents for ablating neuroinflammatory disease.

Cannabinoids have been reported to alter the activities of immune cells in vitro and in vivo. These compounds may serve as ideal agents for adjunct treatment of pathological processes that have a neuroinflammatory component. As highly lipophilic molecules, they readily access the brain. Furthermore, they have relatively low toxicity and can be engineered to selectively target cannabinoid receptors. To date, two cannabinoid receptors have been identified, characterized and designated CB(1) and CB(2). CB(1) appears to be constitutively expressed within the CNS while CB(2) apparently is induced during inflammation. The inducible nature of expression of CB(2) extends to microglia, the resident macrophages of the brain that play a critical role during early stages of inflammation in that compartment. Thus, the cannabinoid-cannabinoid receptor system may prove therapeutically manageable in ablating neuropathogenic disorders such as Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, HIV encephalitis, closed head injury, and granulomatous amebic encephalitis.

HIV-1 Tat and morphine have interactive effects on oligodendrocyte survival and morphology.

Human immunodeficiency virus (HIV)-infected individuals who abuse opiates show faster progression to AIDS, and enhanced incidence of HIV-1 encephalitis. Most opiates with abuse liability are preferential agonists for mu-opioid receptors (MORs), and MORs are expressed on both neurons and glia, including oligodendrocytes (OLs). Tat, gp120, and other viral toxins, cause neurotoxicity in vitro and/or when injected into brain, and co-exposure to opiates can augment HIV-1 protein-induced insults to both glial and neuronal populations. We examined the effects of HIV-1 Tat +/- opiate exposure on OL survival and differentiation. In vivo studies utilized transgenic mice expressing Tat(1-86) regulated by an inducible glial fibrillary acidic protein promoter. Although MBP levels were unchanged on immunoblots, certain structural and apoptotic indices were abnormal. After only 2 days of Tat induction, OLs showed an upregulation of active caspase-3 that was enhanced by morphine exposure. Tat also upregulated TUNEL staining, but only in the presence of morphine. Tat significantly reduced the length of processes in Golgi-Kopsch impregnated OLs. A greater proportion of cells exhibited diminished or aberrant cytoplasmic processes, especially when mice expressing Tat were co-exposed to morphine. Collectively, our data show that OLs in situ are extremely sensitive to effects of Tat +/- morphine, although it is not clear if immature OLs as well as differentiated OLs are targeted equally. Significant elevations in caspase-3 activity and TUNEL labeling, and evidence of increased degeneration/regeneration of OLs exposed to Tat +/- morphine suggest that toxicity toward OLs may be accompanied by heightened OL turnover.

Peroxisome proliferator-activated receptor-γ activation suppresses HIV-1 replication in an animal model of encephalitis

Poor penetration of antiretroviral therapy across the blood-brain barrier poses an impediment on control of HIV-1 infection in brain macrophages. Peroxisome proliferator-activated receptor (PPAR)-gamma, a member of the nuclear receptors family, regulates important physiological functions (including anti-inflammatory effects) in response to ligand-mediated activation. As PPARgamma agonists are rapidly absorbed by oral administration and efficiently permeate the blood-brain barrier, we hypothesized that PPARgamma stimulation may suppress HIV-1 replication. We investigated the effect of PPARgamma ligand (rosiglitazone) on HIV-1 replication in human monocyte-derived macrophages and in vivo using a murine model (immunodeficient mice reconstituted with human lymphocytes and intracerebrally inoculated with HIV-1 infected macrophages) of HIV-1 encephalitis. Treatment with rosiglitazone caused a significant decrease of virus infection in macrophages. PPARgamma stimulation inhibited virus replication by modulating NF-kappaB activation in a receptor-dependent manner, leading to downregulation of HIV-1 long terminal repeat (LTR) promoter activity and suppression of HIV-1 replication. These effects were PPARgamma specific as PPARgamma silencing or addition of PPARgamma antagonist abolished effects of PPARgamma stimulation on HIV-1 LTR and virus replication. Using a murine model for HIV-1 encephalitis, we demonstrated that PPARgamma ligand suppressed HIV-1 replication in macrophages in brain tissue and reduced viremia by 50%. In vitro data delineated the novel mechanism by which PPARgamma activation suppresses HIV-1 replication, and in vivo findings underscored the ability of PPARgamma agonists to reduce HIV-1 replication in lymphocytes and brain macrophages, thus offering a new therapeutic intervention in brain and systemic infection.

Immune Reconstitution Inflammatory Syndrome of the Brain

Immune reconstitution inflammatory syndrome represents a spectrum of clinicopathologic entities encountered in human immunodeficiency virus-infected patients who have received highly active anti-retroviral therapy. The diagnosis is often challenging, treatment options are limited, and the prognosis is variable. To increase awareness and define the clinicopathologic features, we present our experience with 6 probable cases involving the brain, including 1 autopsy. Clinicopathologic review was supplemented by immunohistochemical analysis. There were 5 men and 1 woman, ranging in age from 34 to 47 (mean, 41; SD, 5.39) years. All patients experienced neurologic deterioration (focal deficits in 5/6) after highly active anti-retroviral therapy. All specimens showed a predominance of CD8+ lymphocytic inflammation. Concurrent CNS infections included human immunodeficiency virus encephalitis, progressive multifocal leukoencephalopathy, cryptococcal meningitis, and syphilis. One patient died, 1 was lost to follow-up, 2 improved, and 2 showed no substantial clinical changes. Subtle and overlapping features may preclude a definitive diagnosis. To capture all suspected cases, it is important to consider the possibility of this entity. In this study, the degree of CD8+ inflammation was more pronounced in the single fatal example, and mast cells were not identified in the infiltrates. Although nonspecific, imaging findings may offer clues to early diagnosis.

Ingress of blood-borne macrophages across the blood-brain barrier in murine HIV-1 encephalitis

Blood-borne macrophage ingress into brain in HIV-1 associated neurocognitive disorders governs the tempo of disease. We used superparamagnetic iron-oxide particles loaded into murine bone marrow-derived macrophages (BMM) injected intravenously into HIV-1 encephalitis mice to quantitatively assess BMM entry into diseased brain regions. Magnetic resonance imaging tests were validated by histological coregistration and enhanced image processing. The demonstration of robust BMM migration into areas of focal encephalitis provide ‘proof of concept’ for the use of MRI to monitor macrophage ingress into brain.

Regulation of ABC membrane transporters in glial cells: Relevance to the pharmacotherapy of brain HIV‐1 infection

Limited drug penetration is an obstacle that is often encountered in the treatment of CNS diseases including human immunodeficiency virus type-1 (HIV-1) encephalitis (HIVE). One mechanism that may contribute to this phenomenon is the expression of ATP-binding cassette (ABC) drug efflux transporters [i.e., P-glycoprotein (P-gp), Multidrug Resistance-Associated Proteins (MRP/Mrp), Breast Cancer Resistance Protein (BCRP; also known as ABCG2)] at the primary brain barrier sites (i.e., blood-brain barrier, blood-cerebrospinal fluid barrier). In addition, it has been recently proposed that glial cells may also contribute to the low accumulation and altered distribution of therapeutic compounds in the CNS by functioning as a "secondary barrier." In fact, a few studies have shown that ABC transporters are both expressed and functional in glial cells. Furthermore, commonly prescribed antiretroviral compounds (ARVs), particularly HIV-1 protease inhibitors, are substrates for many of these same transport proteins suggesting that ABC transporters in glial cells may contribute to the overall export of these drugs from the brain. HIV-1 infection is a chronic condition characterized by long-term exposure of brain cellular compartments to HIV-1 virions and soluble viral proteins. In addition, treatment of HIV-1 infection involves long-term administration of a multiplicity of ARVs (i.e., HAART regimens). Indeed, pathological factors associated with HIV-1 infection and/or pharmacological factors related to treatment may alter the expression of ABC transporters and lead to changes in CNS ARV uptake and/or distribution. This review summarizes recent knowledge in this area and emphasizes the role that glial ABC transporters may play in regulating ARV transport.

HIV‐1 viral envelope glycoprotein gp120 produces oxidative stress and regulates the functional expression of multidrug resistance protein‐1 (Mrp1) in glial cells

Brain human immunodeficiency virus type-1 (HIV-1) infection is associated with oxidative stress, which may lead to HIV-1 encephalitis, a chronic neurodegenerative condition. In vitro, oxidative stress can be induced in glial cells by exposure to HIV-1 envelope protein glycoprotein (gp120). Multidrug resistance proteins (Mrps) are known to efflux endogenous substrates (i.e. GSH and GSSG) involved in cellular defense against oxidative stress. Altered GSH/GSSG export may contribute to oxidative damage during HIV-1 encephalitis. At present, it is unknown if gp120 exposure can alter the functional expression of Mrp isoforms. Heat-shock protein 70, inducible nitric oxide synthase, intracellular GSSG, 2',7'-dichlorofluorescein fluorescence, and extracellular nitrite were increased in primary cultures of rat astrocytes triggered with gp120, suggesting an oxidative stress response. RT-PCR and immunoblot analysis demonstrated increased Mrp1 mRNA (2.3-fold) and protein (2.2-fold), respectively, in gp120 treated astrocytes while Mrp4 mRNA or protein expression was not changed. Cellular retention of 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, an established Mrp substrate, was reduced (twofold) in gp120-treated astrocytes, suggesting increased Mrp-mediated transport. In addition, GSH and GSSG export were enhanced in gp120-triggered cells. These data suggest that gp120 can up-regulate Mrp1, but not Mrp4, functional expression in cultured astrocytes. Our observation of increased GSH/GSSG efflux in response to gp120 treatment implies that Mrp isoforms may be involved in regulating the oxidative stress response in glial cells.

Successful Application of Hyperbranched Multidisplacement Genomic Amplification to Detect HIV-1 Sequences in Single Neurons Removed from Autopsy Brain Sections by Laser Capture Microdissection

To confirm studies suggesting that HIV-1 infects neurons and to determine whether CD8(+) T lymphocytes traffic to HIV-1-infected neurons, we used laser capture microdissection to remove hippocampal neurons with and without perineuronal CD8(+) T cells from AIDS patients with HIV-1 encephalitis (HIVE) or without HIVE and from normal controls. We used hyperbranched multidisplacement amplification for whole gene amplification (MDA-WGA) plus two rounds of PCR to amplify housekeeping sequences (HK(+)) and, in HK(+) samples, to amplify HIV-1 gag, nef, and pol sequences. Sample size and, in single neurons, MDA-WGA correlated with housekeeping gene amplification (P < 0.05), whereas patient group and postmortem interval did not (P > 0.05). Neuronal viral sequences correlated with HIVE (43% vs. 13% and 0 in non-HIVE and controls, respectively) and, in HIVE cases, with perineuronal CD8(+) T lymphocytes (70% in CD8(+) samples vs. 37% of CD8(-) samples). Our results suggest that MDA-WGA is a useful technique when analyzing DNA from single cells from autopsy brains, supporting prior studies that show that neurons may contain HIV-1 neuronal sequences in vivo. The association between neuronal infection and perineuronal CD8(+) T cells supports our hypothesis that these cells specifically traffic to infected neurons but raises the possibility that CD8(+) T cells, if infected, could transmit virus to neurons.

YKL-40, a Marker of Simian Immunodeficiency Virus Encephalitis, Modulates the Biological Activity of Basic Fibroblast Growth Factor

Human immunodeficiency virus encephalitis causes dementia in acquired immune deficiency syndrome patients. Using proteomic analysis of postmortem cerebrospinal fluid (CSF) and brain tissue from the simian immunodeficiency virus primate model, we demonstrate here a specific increase in YKL-40 that was tightly associated with lentiviral encephalitis. Longitudinal analysis of CSF from simian immunodeficiency virus-infected pigtailed macaques showed an increase in YKL-40 concentration 2 to 8 weeks before death from encephalitis. This increase in YKL-40 correlated with an increase in CSF viral load; it may therefore represent a biomarker for the development of encephalitis. Analysis of banked human CSF from human immunodeficiency virus-infected patients also demonstrated a correlation between YKL-40 concentration and CSF viral load. In vitro studies demonstrated increased YKL-40 expression and secretion by macrophages and microglia but not by neurons or astrocytes. We found that YKL40 displaced extracellular matrix-bound basic fibroblast growth factor (bFGF) as well as inhibited the mitogenic activity of both fibroblast growth factor receptor 1-expressing BaF3 cells and bFGF-induced axonal branching in hippocampal cultures. Taken together, these findings demonstrate that during lentiviral encephalitis, YKL-40 may interfere with the biological activity of bFGF and potentially of other heparin-binding growth factors and chemokines that can affect neuronal function or survival.

YKL-40: A Candidate Biomarker for Simian Immunodeficiency Virus and Human Immunodeficiency Virus Encephalitis?

This Commentary explores the possible role for YKL-40, a secreted mammalian chitinase-like protein, as a predictive biomarker for SIV encephalitis (SIVE) and HIV encephalitis (HIVE).

Morphine causes rapid increases in glial activation and neuronal injury in the striatum of inducible HIV‐1 tat transgenic mice

HIV encephalitis (HIVE) is accompanied by brain inflammation, leukocyte infiltration, and glial activation, and HIV patients who abuse opiates are more likely to develop HIVE. To better understand how opiates could alter HIV-related brain inflammation, the expression of astrocyte (GFAP immunoreactivity) and macrophage/microglial (F4/80 or Mac1 immunoreactivity) markers in the striatum, and the percentage of 3-nitrotyrosine (3-NT) positive macrophages/microglia, was determined following a 2-day exposure to morphine (5 mg/kg/day via time-release, subcutaneous implant) and doxycycline in GFAP-driven, doxycycline-inducible HIV-1 Tat transgenic mice. Data show that both morphine and Tat induction via doxycycline increased astrocyte activation, with significant additive increases achieved with combined morphine and doxycycline exposure. By contrast, combined Tat induction and morphine exposure, but neither manipulation alone, significantly increased the proportion of macrophages/microglia present in the striatum of transgenic mice, although morphine exposure was necessary to elevate 3-NT co-detection in Mac1-positive macrophages/microglia. Finally, Tat induction increased the percentage of neurons expressing active caspase-3, and this was even more significantly elevated by co-administration of morphine. In spite of elevations in caspase-3, neuronal TUNEL reactivity was unchanged in all groups, even after 10 days of Tat induction. Importantly, co-administration of naltrexone completely antagonized the effects of morphine. These findings indicate that morphine rapidly and significantly increases the activation of astrocytes and macrophages/microglia in the brains of inducible Tat transgenic mice, supporting the theory that early inflammatory changes in glia could underlie the development of HIVE in opiate-abusing AIDS patients.

Longitudinal in Vivo Positron Emission Tomography Imaging of Infected and Activated Brain Macrophages in a Macaque Model of Human Immunodeficiency Virus Encephalitis Correlates with Central and Peripheral Markers of Encephalitis and Areas of Synaptic Degeneration

Human immunodeficiency virus encephalitis is characterized by infiltration of the brain with infected and activated macrophages; however, it is not known why disease occurs after variable lengths of infection in 25% of immunosuppressed acquired immune deficiency syndrome patients. We determined in vivo correlates (in peripheral blood and the central nervous system) for the development and progression of lentiviral encephalitis by longitudinally following infected and activated macrophages in the brain using positron emission tomography (PET). Using human postmortem brain tissues from both lentivirus-infected encephalitic patients and cell culture systems, we showed that the PET ligand [(3)H](R)-PK11195 bound specifically to virus-infected and activated macrophages. We longitudinally imaged infected and activated brain macrophages in a cohort of macaques infected with simian immunodeficiency virus using [(11)C](R)-PK11195. [(11)C](R)-PK11195 retention in vivo in the brain correlated with viral burden in the brain and cerebrospinal fluid, and with regions of both presynaptic and postsynaptic damage. Finally, longitudinal changes in [(11)C](R)-PK11195 retention in the brain in vivo correlated with changes in circulating monocytes as well as in both natural killer and memory CD4(+) T cells in the periphery. Our results suggest that development and progression of simian immunodeficiency virus encephalitis in vivo correlates with changes in specific cell subtypes in the periphery. A combination of PET imaging and the assessment of these peripheral immune parameters may facilitate longitudinal assessment of lentiviral encephalitis in living patients as well as evaluation of therapeutic efficacies.

Short Communication:Absence of HIV Infection in the Choroid Plexus of Two Patients Who Died Rapidly with HIV-Associated Dementia

Absence of HIV infection of the choroid plexus (CPx) and macrophages in choroidal stroma was observed in two HIV-infected individuals who died 7 weeks and 12 months following the onset of HIV encephalitis. In contrast, the profound macrophage-related pathology associated with HIV infection presented in other neural tissue from 48 brain regions (seven CPx) was analyzed. These data suggest that HIV entry to the CNS may be independent of the CPx. It also emphasizes that the CPx is unlikely to harbor a significant reservoir of HIV in patients who rapidly progress to dementia.

Diagnosis of cerebral toxoplasmosis by detection of Toxoplasma gondii tachyzoites in cerebrospinal fluid

Sirs: In patients with human immune deficiency syndrome, cerebral toxoplasmosis is one of the typical opportunistic diseases. Compared to cardiac and pulmonary involvement, cerebral manifestation is more common and mediated by the neurotoxic potential of the HIV [1]. Usually, toxoplasmosis can be diagnosed by detecting organism-specific antibodies (IgG, IgM) in blood samples and cerebrospinal fluid (CSF). However, in immunodeficient patients, in particular those with HIV infection in a progressive stage (CD4+-cell count less than 200), the diagnosis of cerebral toxoplasmosis may cause difficulties if only relying on antibody detection. Other diagnostic options are either less specific, less available or more invasive [2, 3]. In the present report, we detected Toxoplasma gondii (Tg) tachyzoites in lumbar CSF in a patient with cerebral toxoplasmosis. To the best of our knowledge, cytologic detection of Tg tachyzoites in lumbar CSF is described for the first time. A 50-year-old female with a 9 year history of untreated HIV infection (CD4+-T-lymphocytes 210/μL corresponding to stage C2 of the CDC 1993 criteria, and HIV viral load more than 100,000 HIVRNA-Genome/ml) with rapid progressive symptoms of encephalitis was admitted to our hospital. The patient was somnolent and disoriented, and she showed signs of basal meningitis consisting of paresis of right-sided cranial nerves VI and VII. In the MRI scan multiple and disseminated T2-lesions were found in supraand infratentorial regions, in particular in the brainstem and cerebellum, where lesions showed ring-shaped contrast enhancement. Meningeal enhancement was described as well. Apart from the MRI results suggestive of opportunistic infection, CSF findings showed a pleocytosis (66 leukocytes/μl), elevation of total protein (1474 mg/dl) and accordingly a moderate dysfunction of the blood-CSF barrier (CSF/serum quotient of albumin 24.0 10–3, ref.-range < 8.0 10–3). The differential cell count of the CSF revealed 87 % lymphocytes, 3 % neutrophils, 1 % eosinophils, 5 % plasma cells and the remaining 4 % were apoptotic cells. There was no evidence for infection with Cryptococcus. Serum anti-Tg IgM-antibodies were within the normal range (normal range < 20 U/L) and IgG-antibodies were slightly increased (113 U/L, normal range < 90 U/L). In CSF antibodies against Tg were not elevated. However, interpretation of these results were difficult given the immune deficient condition of the patient. CSF cytology, however, revealed the presence of mostly intracellular organisms (small bow-shaped organisms with slightly eccentrically placed nuclei) morphologically suggestive as Tg tachyzoites and bradyzoites (Fig. 1) in accordance with the previous literature [4]. These findings were confirmed by post-mortem examination of brain tissue. The autopsy revealed the diagnosis of disseminated focal necrotizing Tg encephalitis involving the cerebrum, cerebellum and brainstem. In addition to inflammatory changes typical for HIV encephalitis, Tg organisms were described in multiple brain regions confirmed by immunohistochemical staining. Leptomeningeal infiltration was also found in several regions primarily in parasagittal and right-sided prefrontal regions. Overall the neuropathology findings were in accordance with the MRI findings. The patient refused antiviral as well as anti-toxoplasma treatment, only support measures were provided owing to the patient’s request. She died two weeks after admission to the hospital and 9 years after she was diagnosed with HIV. A possible way to detect Tg organisms is a direct cytologic examination of CSF. However, this procedure is associated with a low sensitivity as described in a large retrospective study where tachyzoites were seen in only 2 out of 6,090 examined CSF’s both of which were ventricular specimens [4]. So far, only five cases exist in the literature where a direct identification of Tg organisms was possible by cytologic examination of CSF [4–8]. However, in all reported cases Tg organisms were detected only in ventricular CSF but not in lumbar CSF. Therefore, it was discussed that lack of detection of tachyzoites in lumbar CSF was due to obstructive hydrocephalus inhibiting the passover of tachyzoites from the ventricles to the subarachnoid space [3]. In our patient a hydroLETTER TO THE EDITORS