Kenneth L. Tyler

HAART improves prognosis in HIV-associated progressive multifocal leukoencephalopathy

Article abstract Introduction of highly active antiretroviral therapy (HAART) has been associated with many changes in the complications of human immunodeficiency virus (HIV) infection. A cohort of 25 HIV patients with progressive multifocal leukoencephalopathy (PML) treated with HAART experienced a median survival of >46 weeks. This is an improvement in prognosis compared with recent historic experience and correlated with HIV RNA viral load reductions. We conclude that current HIV therapy is important in improving the outlook of PML in the setting of HIV.

West Nile virus neuroinvasive disease

Since 1999, there have been nearly 20,000 cases of confirmed symptomatic West Nile virus (WNV) infection in the United States, and it is likely that more than 1 million people have been infected by the virus. WNV is now the most common cause of epidemic viral encephalitis in the United States, and it will likely remain an important cause of neurological disease for the foreseeable future. Clinical syndromes produced by WNV infection include asymptomatic infection, West Nile Fever, and West Nile neuroinvasive disease (WNND). WNND includes syndromes of meningitis, encephalitis, and acute flaccid paralysis/poliomyelitis. The clinical, laboratory, and diagnostic features of these syndromes are reviewed here. Many patients with WNND have normal neuroimaging studies, but abnormalities may be present in areas including the basal ganglia, thalamus, cerebellum, and brainstem. Cerebrospinal fluid invariably shows a pleocytosis, with a predominance of neutrophils in up to half the patients. Diagnosis of WNND depends predominantly on demonstration of WNV‐specific IgM antibodies in cerebrospinal fluid. Recent studies suggest that some WNV‐infected patients have persistent WNV IgM serum and/or cerebrospinal fluid antibody responses, and this may require revision of current serodiagnostic criteria. Although there is no proven therapy for WNND, several vaccines and antiviral therapy with antibodies, antisense oligonucleotides, and interferon preparations are currently undergoing human clinical trials. Recovery from neurological sequelae of WNV infection including cognitive deficits and weakness may be prolonged and incomplete. Ann Neurol 2006;60:286–300

Rituximab-associated progressive multifocal leukoencephalopathy in rheumatoid arthritis.

OBJECTIVE To describe the development of progressive multifocal leukoencephalopathy (PML) in patients with rheumatoid arthritis (RA) treated with rituximab. DESIGN Case study. SETTING Clinical care for patients with rheumatologic diseases. Most were referred to academic centers for care after diagnosis (Washington University, St Louis, Missouri; Karolinska Insitute, Stockholm, Sweden; and Royal Melbourne Hospital, Melbourne, Australia) while one was cared for in a neurology practice in Dallas, Texas, with consultation by an academic neurovirologist from the University of Colorado in Denver. PATIENTS Four patients developing PML in the setting of rituximab therapy for RA. INTERVENTION Rituximab therapy. MAIN OUTCOME MEASURES Clinical and pathological observations. RESULTS Four patients from an estimated population of 129 000 exposed to rituximab therapy for RA are reported in whom PML developed after administration of this drug. All were women older than 50 years, commonly with Sjögren syndrome and a history of treatment for joint disease ranging from 3 to 14 years. One case had no prior biologic and minimal immunosuppressive therapy. Progressive multifocal leukoencephalopathy presented as a progressive neurological disorder, with diagnosis confirmed by detection of JC virus DNA in the cerebrospinal fluid or brain biopsy specimen. Two patients died in less than 1 year from PML diagnosis, while 2 remain alive after treatment withdrawal. Magnetic resonance scans and tissue evaluation confirmed the frequent development of inflammatory PML during the course of the disease. CONCLUSION These cases suggest an increased risk, about 1 case per 25 000 individuals, of PML in patients with RA being treated with rituximab. Inflammatory PML may occur in this setting even while CD20 counts remain low.

Reovirus-Induced Apoptosis Is Mediated by TRAIL

ABSTRACT Members of the tumor necrosis factor (TNF) receptor superfamily and their activating ligands transmit apoptotic signals in a variety of systems. We now show that the binding of TNF-related, apoptosis-inducing ligand (TRAIL) to its cellular receptors DR5 (TRAILR2) and DR4 (TRAILR1) mediates reovirus-induced apoptosis. Anti-TRAIL antibody and soluble TRAIL receptors block reovirus-induced apoptosis by preventing TRAIL-receptor binding. In addition, reovirus induces both TRAIL release and an increase in the expression of DR5 and DR4 in infected cells. Reovirus-induced apoptosis is also blocked following inhibition of the death receptor-associated, apoptosis-inducing molecules FADD (for FAS-associated death domain) and caspase 8. We propose that reovirus infection promotes apoptosis via the expression of DR5 and the release of TRAIL from infected cells. Virus-induced regulation of the TRAIL apoptotic pathway defines a novel mechanism for virus-induced apoptosis.

Progressive Esophagitis from Acyclovir-Resistant Herpes Simplex: Clinical Roles for DNA Polymerase Mutants and Viral Heterogeneity?

Clinically acquired acyclovir resistance in herpes simplex has usually been associated with a deficiency in viral thymidine kinase, which, in turn, has been linked with attenuated virulence in animal models. Diminished pathogenicity in thymidine kinase-deficient isolates has been partly responsible for controversies about the clinical significance of antiviral resistance. We report on a series of resistant virus isolates from a patient who had severe, progressive esophagitis. These isolates had various thymidine kinase activities, ranging from 2.8% to 130% when compared with the activity of the isolate obtained before treatment; the resistant isolate 615 retained enzyme activity as well as neurovirulence in an encephalitis model. Plaque purification showed a heterogeneous mixture containing at least one acyclovir-resistant, foscarnet-resistant plaque isolate (615.8) fully able to phosphorylate acyclovir. The 3.3-kbp BamHI fragment containing most of the DNA polymerase gene from isolate 615.8 was purified and used to successfully transfer both acyclovir and foscarnet resistance. Acquisition of in-vitro acyclovir resistance was associated with progression of clinical disease, as well as with maintenance of pathogenicity in an animal model and at least one mutation in viral DNA polymerase. Patients with herpes simplex infections that progress during acyclovir therapy should be observed for acquisition of resistance in the setting of antiviral chemotherapy; future studies should also consider the presence of heterogeneous virus populations in such patients.

Molecular Methods for Diagnosis of Viral Encephalitis

SUMMARY Hundreds of viruses cause central nervous system (CNS) disease, including meningoencephalitis and postinfectious encephalomyelitis, in humans. The cerebrospinal fluid (CSF) is abnormal in >90% of cases; however, routine CSF studies only rarely lead to identification of a specific etiologic agent. Diagnosis of viral infections of the CNS has been revolutionized by the advent of new molecular diagnostic technologies to amplify viral nucleic acid from CSF, including PCR, nucleic acid sequence-based amplification, and branched-DNA assay. PCR is ideally suited for identifying fastidious organisms that may be difficult or impossible to culture and has been widely applied for detection of both DNA and RNA viruses in CSF. The technique can be performed rapidly and inexpensively and has become an integral component of diagnostic medical practice in the United States and other developed countries. In addition to its use for identification of etiologic agents of CNS disease in the clinical setting, PCR has also been used to quantitate viral load and monitor duration and adequacy of antiviral drug therapy. PCR has also been applied in the research setting to help discriminate active versus postinfectious immune-mediate disease, identify determinants of drug resistance, and investigate the etiology of neurologic disease of uncertain cause. This review discusses general principles of PCR and reverse transcription-PCR, including qualitative, quantitative, and multiplex techniques, with comment on issues of sensitivity, specificity, and positive and negative predictive values. The application of molecular diagnostic methods for diagnosis of specific infectious entities is reviewed in detail, including viruses for which PCR is of proven efficacy and is widely available, viruses for which PCR is less widely available or for which PCR has unproven sensitivity and specificity, and nonviral entities which can mimic viral CNS disease.

Distinct pathways of viral spread in the host determined by reovirus S1 gene segment

The genetic and molecular mechanisms that determine the capacity of a virus to utilize distinct pathways of spread in an infected host were examined by using reoviruses. Both reovirus type 1 and reovirus type 3 spread to the spinal cord following inoculation into the hindlimb or forelimb footpad of newborn mice. For type 3 this spread is through nerves and occurs via the microtubule-associated system of fast axonal transport. By contrast, type 1 spreads to the spinal cord through the bloodstream. With the use of reassortant viruses containing various combinations of double-stranded RNA segments (genes) derived from type 1 and type 3, the viral S1 double-stranded RNA segment was shown to be responsible for determining the capacity of reoviruses to spread to the central nervous system through these distinct pathways.

Reovirus-Induced Apoptosis Requires Activation of Transcription Factor NF-κB

ABSTRACT Reovirus infection induces apoptosis in cultured cells and in vivo. To identify host cell factors that mediate this response, we investigated whether reovirus infection alters the activation state of the transcription factor nuclear factor kappa B (NF-κB). As determined in electrophoretic mobility shift assays, reovirus infection of HeLa cells leads to nuclear translocation of NF-κB complexes containing Rel family members p50 and p65. Reovirus-induced activation of NF-κB DNA-binding activity correlated with the onset of NF-κB-directed transcription in reporter gene assays. Three independent lines of evidence indicate that this functional form of NF-κB is required for reovirus-induced apoptosis. First, treatment of reovirus-infected HeLa cells with a proteasome inhibitor prevents NF-κB activation following infection and substantially diminishes reovirus-induced apoptosis. Second, transient expression of a dominant-negative form of IκB that constitutively represses NF-κB activation significantly reduces levels of apoptosis triggered by reovirus infection. Third, mutant cell lines deficient for either the p50 or p65 subunits of NF-κB are resistant to reovirus-induced apoptosis compared with cells expressing an intact NF-κB signaling pathway. These findings indicate that NF-κB plays a significant role in the mechanism by which reovirus induces apoptosis in susceptible host cells.

Herpes simplex virus infection as a cause of benign recurrent lymphocytic meningitis

Table. SI Units Herpes simplex viruses (HSV) type 1 and type 2 establish latent infections in the peripheral nervous system of humans [1, 2]. Reactivation of latent HSV infection from sensory ganglia results in a broad range of clinical manifestations, depending on the site of latency, virus type, and immune competency of the host. The major central nervous system consequences of HSV reactivation are encephalitis and meningitis [3-5]. Meningitis and encephalitis caused by HSV infection may be difficult to recognize because they are only rarely associated with clinical evidence of extraneural infection [4]. Even when meningitis results from reactivation of latent HSV type 2 infection in patients with known genital herpes, coincident herpetic skin lesions are seldom documented [5, 6]. This absence of extraneural manifestations increases the importance of detecting HSV in cerebrospinal fluid or brain tissue or both. Viral cultures rarely show HSV type 1 in cerebrospinal fluid during viral encephalitis [4]. Herpes simplex virus type 2 is more commonly cultured from cerebrospinal fluid during meningitis associated with the first episode of genital HSV type 2 infection but is rarely cultured during meningitis associated with recurrent genital herpes [5, 7-9]. The failure of culture techniques has led to the use of new diagnostic techniques. These include the detection of antibodies to HSV in cerebrospinal fluid by immunoblot analysis [10] or enzyme-linked immunosorbent assay [5, 11] and the detection of HSV DNA using the polymerase chain reaction (PCR) [12-17]. The PCR assay is extremely sensitive and highly specific for the diagnosis of HSV infections of the central nervous system [12-17]. With a single exception [18], it has not been possible to isolate HSV from cerebrospinal fluid of patients with recurrent meningitis [5-9]. However, HSV DNA has been found in samples of cerebrospinal fluid from isolated patients with benign recurrent lymphocytic meningitis [16, 19, 20]. Therefore, we used PCR DNA amplification and type-specific antibody detection in cerebrospinal fluid samples to investigate the role of HSV in a large number of patients with the syndrome of benign, recurrent, self-limited aseptic meningitis [21]. Methods Specimen Preparation and Polymerase Chain Reaction Specimens submitted for PCR analysis for HSV were subjected to precautionary procedures to avoid contaminating the samples and the DNA amplification reactions with exogenous DNA [22]. The PCR assay for HSV was done as described previously [16] with minor modifications. Specimens were analyzed shortly after receipt by the laboratory. They were treated with the chelating InstaGene DNA purification matrix (BioRad Laboratories, Inc.; Hercules, California) before amplification. Briefly, the aqueous volume of the InstaGene slurry was first decreased by one half; to 100 L of the decreased slurry, 20 L of cerebrospinal fluid was added, and incubation steps were carried out according to the manufacturers guidelines. Polymerase chain reaction mixtures contained 20 L of InstaGene-treated cerebrospinal fluid in a total volume of 50 L. Each reaction contained 1X PfuI buffer, 200 mol/L each of four deoxynucleoside triphosphates, 1.25 units of PfuI (Stratagene; La Jolla, California), and 0.8 mol/L of each primer, HSVPolA1 and HSVPolA2 [16]. The samples were amplified for 45 cycles. All DNA amplification experiments included a negative water control and a positive sensitivity control, which consisted of 20 molecules of the plasmid pGX146. This plasmid contains the Kpn I x fragment of the HSV type 1 strain 17 DNA polymerase gene [23]. Appropriate clinical controls to compare with our test specimens cannot be obtained. In order to verify the specificity of the DNA amplification procedure, we used negative control specimens from patients with neurologic diseases who did not have herpes as specified (Table 1, patients 1 to 6) and used positive control specimens from patients with herpes simplex infections who were diagnosed by an alternate method (Table 1, patients 7 to 13). Experiments for DNA amplification were prepared in parallel with blindly coded cerebrospinal fluid specimens. Cerebrospinal fluid specimens from patients with benign recurrent lymphocytic meningitis were tested in parallel with one positive control specimen of cerebrospinal fluid (Table 1), in addition to the pGX146 and water controls. To test for the presence of PCR inhibitors in specimens that yielded negative results in DNA amplification experiments, reconstruction experiments were done in which InstaGene DNA preparations were spiked with 20 molecules of pGX146 and subjected to amplification. Restriction digests of DNA amplification products were typed, as previously described [16]. Table 1. Selected Clinical Data for Control Cerebrospinal Fluid Specimens* Detection of Anti-Herpes Simplex Virus Antibodies Anti-HSV antibodies were detected as previously described [16, 24, 25]. Results were scored as positive for HSV type 1 if a predominance of antibodies to HSV type 1 (at least four bands) was apparent and if no antibodies to HSV type 2 gG were detected. Results were scored as positive for HSV type 2 if a predominance of antibodies to HSV type 2 (at least four bands) was detected including a band corresponding to HSV type 2 gG. Herpes simplex virus type 2 atypical profiles consisted of four or more HSV type 2 bands and a lack of detectable antibody to HSV type 2 gG. Such profiles are apparent in 8% to 10% of patients with culture-documented HSV type 2 genital infections [25, 26]. Weak profiles refer to those that are scored using a 1:25 dilution of cerebrospinal fluid rather than the usual 1:50 dilution. Results Between November 1990 and 15 June 1993, 156 cerebrospinal fluid specimens were received for PCR analysis for HSV. Clinical information accompanying these specimens was often limited. Approximately 80% of specimens were submitted because of presumed encephalitis or encephalopathic syndrome, 10% for presumed neonatal herpes simplex infection, 5% for myelitis or neuropathy or both, and 5% for miscellaneous diagnoses. Twenty of the cerebrospinal fluid specimens were from patients with a presumptive diagnosis of recurrent viral meningitis. The specimens were submitted directly or shipped on dry ice by overnight mail to the Diagnostic Virology Laboratory of the University of Colorado Health Sciences Center between November 1990 and 15 June 1993. Thirteen of these 20 [65%] specimens analyzed for HSV DNA were obtained during the acute phase of the illness, were not previously exposed to prolonged thermal trauma, and met the following preestablished diagnostic criteria: 1) at least three episodes of meningitis occurring in the absence of other clinically significant neurologic signs and symptoms that resolved spontaneously without residual sequelae; 2) predominant cerebrospinal fluid lymphocytic pleocytosis; 3) negative or normal test results after culturing cerebrospinal fluid specimens for viruses, bacteria, Mycobacterium tuberculosis, and fungi; normal cytologic appearance; and the absence of treponemal or cryptococcal antigens; 4) no signs or symptoms of a uveomeningitic syndrome on routine ophthalmologic examination; 5) no evidence by history, examination, or laboratory tests to suggest the presence of collagen vascular disease, vasculitis, or drug-related hypersensitivity meningitis; 6) no evidence by computed tomography or magnetic resonance imaging [or both] of intracranial mass lesions; and 7) no evidence of herpetic skin lesions. Seven of the 20 submitted samples from patients were excluded for the following reasons: polymorphonuclear pleocytosis (3 patients), fewer than three attacks of meningitis (1 patient), systemic lupus erythematosus (1 patient), convalescent specimen (1 patient), and inadequate specimen handling (1 patient). Clinical information was obtained from chart review and from the referring physicians. Genital HSV cultures and complete pelvic examinations were not done on all patients; therefore, we cannot exclude the possibility that some patients without evident herpetic skin lesions were asymptomatically shedding HSV from the genital tract. Patients who previously had herpetic skin lesions were not excluded but were considered as a separate group from those meeting all seven criteria. Of the 13 patients (Table 2) evaluated in this study, 9 were women. Patients had 3 to 9 attacks of meningitis (mean, 4.6 attacks) during a time interval of 2 to 21 years (mean, 8.4 years). Attacks of meningitis lasted 3 to 14 days (mean, 6.3 days). Cerebrospinal fluid specimens contained 48 to 1600 cells/mm3 (mean, 443 cells/mm3) with 58% to 98% lymphocytes. In the samples of cerebrospinal fluid, glucose levels were normal and protein levels ranged from 0.41 to 2.4 g/L (41 to 240 mg/dL), with a mean level of 1.22 g/L (122 mg/dL). Table 2. Selected Clinical Data for Study Patients* In 11 patients (84.6%; CI, 55% to 98%), HSV DNA was detected by PCR; the amplified sequence of DNA in 10 of these specimens was HSV type 2. In all of the cerebrospinal fluid specimens with HSV type 2 DNA, HSV type 2 antibody was also present. The cerebrospinal fluid from one patient had HSV type 1 DNA and HSV type 1 antibodies. Herpes simplex virus DNA could not be detected in cerebrospinal fluid specimens from two patients, although both contained antibody to HSV type 2. Thus, only 2 of 13 patients had benign recurrent lymphocytic meningitis that was not associated with an HSV infection in the central nervous system, as defined by the presence of HSV DNA. To ensure that failure to detect HSV DNA in the cerebrospinal fluid from patients 9 and 10 (Table 2) was not caused by the presence of PCR inhibitors, known amounts of pGX146 template DNA were added to specimens and amplification was done. These experiments consistently showed detection of DNA amplification products from an input of as low as 20 molecules

Emerging Viral Infections of the Central Nervous System: Part 1

In this 2-part review, I will focus on emerging virus infections of the central nervous system (CNS). Part 1 will introduce the basic features of emerging infections, including their definition, epidemiology, and the frequency of CNS involvement. Important mechanisms of emergence will be reviewed, including viruses spreading into new host ranges as exemplified by West Nile virus (WNV), Japanese encephalitis (JE) virus, Toscana virus, and enterovirus 71 (EV71). Emerging infections also result from opportunistic spread of viruses into known niches, often resulting from attenuated host resistance to infection. This process is exemplified by transplant-associated cases of viral CNS infection caused by WNV, rabies virus, lymphocytic choriomeningitis, and lymphocytic choriomeningitis-like viruses and by the syndrome of human herpesvirus 6 (HHV6)-associated posttransplantation acute limbic encephalitis. The second part of this review begins with a discussion of JC virus and the occurrence of progressive multifocal leukoencephalopathy in association with novel immunomodulatory therapies and then continues with an overview of the risk of infection introduced by imported animals (eg, monkeypox virus) and examples of emerging diseases caused by enhanced competence of viruses for vectors and the spread of vectors (eg, chikungunya virus) and then concludes with examples of novel viruses causing CNS infection as exemplified by Nipah and Hendra viruses and bat lyssaviruses.