Todd P. Margolis

Unusual Abundance of Atypical Strains Associated with Human Ocular Toxoplasmosis

To facilitate genotyping of Toxoplasma gondii in vitreous fluid of patients with severe or atypical ocular toxoplasmosis, polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) assays were developed for SAG3 (p43) and SAG4 (p18), 2 single-copy surface antigen genes. Together with strategies for SAG1, SAG2, and B1, multilocus RFLP analyses were performed on PCR-amplified parasite DNA present in 12 clinical specimens. Most samples (8/12) were not infected by type II or type III mouse-avirulent strains. Only 1 type III and 3 type II strains were identified, all from immunosuppressed patients. In 6 otherwise healthy adults and in 1 immunosuppressed patient, the SAG1 allele associated with mouse virulence was amplified. Of 12 samples, 3 possessed true type I strains; 5 of 12 had new recombinant genotypes with alleles typical of type I or III strains at all loci examined. The unusual bias toward type I and/or recombinant genotypes bearing the SAG1 type I allele associated with mouse virulence in immunocompetent adults has important implications for the epidemiology and efficacious treatment of ocular toxoplasmosis.

A latent, nonpathogenic HSV-1-derived vector stably expresses β-galactosidase in mouse neurons

A genetically engineered herpes simplex virus variant was constructed for use as a stable gene vector for neurons. To inhibit replication, the agent possessed a deletion in the immediate early gene ICP4, and to minimize reactivation from the latent state, the gene encoding the latency-associated transcript was deleted. The E. coli beta-galactosidase gene under the control of the Maloney murine leukemia virus long terminal repeat promoter was inserted into the ICP4 region. When introduced into the peripheral nervous system, this virus established latent infections and stably expressed beta-galactosidase in primary sensory neurons. Expression of beta-galactosidase over a more limited time period was observed when the latent infection was established in motor neurons of the hypoglossal nucleus. Agents of this general design have considerable potential for use as gene vectors for studies of neuronal function and correction of genetic defects affecting neurons.

The progressive outer retinal necrosis syndrome. A variant of necrotizing herpetic retinopathy in patients with AIDS.

BACKGROUND The progressive outer retinal necrosis syndrome is a recently recognized variant of necrotizing herpetic retinopathy. This report characterizes more fully its clinical features and course. METHODS Using standardized clinical criteria, patients with progressive outer retinal necrosis syndrome from four institutions were identified. Patient records were reviewed retrospectively for the following data: medical and demographic characteristics, presenting symptoms, physical findings, course, responses to treatment, and outcomes. RESULTS Thirty-eight patients (65 involved eyes) were studied. All had acquired immune deficiency syndrome. A known history of cutaneous zoster was documented in 22 (67%) of 33 patients. Median CD4 lymphocyte count was 21/mm3 (range, 0-130/mm3). Median follow-up was 12 weeks. The most common presenting symptom was unilateral decreased vision (35 of 65 eyes, 54%); median visual acuity at presentation was 20/30 (range, 20/20 to no light perception [NLP]). Anterior chamber and vitreous inflammatory reactions were absent or minimal in all patients. Typical retinal lesions were multifocal, deep opacities scattered throughout the periphery, although macular lesions also were present in 21 eyes (32%) at diagnosis. Lesions progressed rapidly to confluence. Initial intravenous antiviral therapy appeared to reduce disease activity in 17 (53%) of 32 eyes, but treatment did not alter final visual outcome. Visual acuity was NLP in 42 (67%) of 63 eyes within 4 weeks after diagnosis. Retinal detachment occurred in 43 (70%) of 61 eyes, including 13 (93%) of 14 eyes that received prophylactic laser retinopexy. CONCLUSION The progressive outer retinal necrosis syndrome is characterized by features that distinguish it from cytomegalovirus retinopathy, acute retinal necrosis syndrome, and other necrotizing herpetic retinopathies. Visual prognosis is poor with current therapies.

Spontaneous molecular reactivation of herpes simplex virus type 1 latency in mice

Infection of the mouse trigeminal ganglia (TG) is the most commonly used model for the study of herpes simplex virus type 1 (HSV-1) latency. Its popularity is caused, at least in part, by the perception that latent infection can be studied in this system in the absence of spontaneous viral reactivation. However, this perception has never been rigorously tested. To carefully study this issue, the eyes of Swiss–Webster mice were inoculated with HSV-1 (KOS), and 37–47 days later the TG were dissected, serial-sectioned, and probed for HSV-1 ICP4, thymidine kinase, glycoprotein C, and latency-associated transcript RNA by in situ hybridization. Serial sections of additional latently infected TG were probed with HSV-1-specific polyclonal antisera. Analysis of thousands of probed sections revealed abundant expression of viral transcripts, viral protein, and viral DNA replication in about 1 neuron per 10 TG tested. These same neurons were surrounded by a focal white cell infiltrate, indicating the presence of an antigenic stimulus. We conclude that productive cycle viral genes are abundantly expressed in rare neurons of latently infected murine TG and that these events are promptly recognized by an active local immune response. In the absence of detectable infectious virus in these ganglia, we propose the term “spontaneous molecular reactivation” to describe this ongoing process.

Transient vitreous inflammatory reactions associated with combination antiretroviral therapy in patients with AIDS and cytomegalovirus retinitis

PURPOSE To report the observation that a transient vitreous inflammatory reaction may develop in the eyes of patients with acquired immunodeficiency syndrome (AIDS), cytomegalovirus retinitis, and an increased CD4+ T-lymphocyte count during treatment with antiretroviral therapy including a protease inhibitor. METHODS We reviewed the medical records of eight patients with AIDS and cytomegalovirus retinitis who developed vitreous inflammatory reactions greater than those usually seen with this disease. RESULTS Vitreous inflammatory reactions obscured the view of the posterior pole in all patients. No iris nodules, synechiae, glaucoma, or cystoid macular edema were observed. Six patients had unilateral cytomegalovirus retinitis, and, in each, the inflammation occurred only in the eye with cytomegalovirus retinitis. The vitreous inflammatory reactions were associated with clinically inactive cytomegalovirus retinitis in six patients, with disease reactivation in one patient, and were present at diagnosis of active disease in one patient. Cytomegalovirus retinitis has not recurred in any of these patients since their episodes of vitreous inflammation. Vitreous inflammation developed in all eight patients after a substantial increase in CD4+ T-lymphocyte counts caused by combination antiretroviral therapy. Five patients had CD4+ T-lymphocyte counts of greater than 100 cells per microl at the time the vitreous inflammatory reaction developed. No other causes of uveitis were found. CONCLUSIONS Patients with AIDS and cytomegalovirus retinitis may develop transient intraocular inflammation associated with combination antiretroviral therapy. We believe that this inflammation reflects an improved immune response against cytomegalovirus.

Herpetic Eye Disease Study: A Controlled Trial of Oral Acyclovir for Herpes Simplex Stromal Keratitis

PURPOSE To evaluate the efficacy of oral acyclovir in treating stromal keratitis caused by herpes simplex virus (HSV) in patients receiving concomitant topical corticosteroids and trifluridine. METHODS The authors performed a randomized, double-masked, placebo-controlled, multicenter trial in 104 patients with HSV stromal keratitis without accompanying HSV epithelial keratitis. Sample size was chosen so that a 5%, one-tailed test would have an 80% chance of detecting a doubling of the median time to treatment failure. Patients were randomized to receive a 10-week course of either oral acyclovir (400 mg 5 times daily, n = 51) or placebo (n = 53). All patients also received a standard regimen of topical prednisolone phosphate and trifluridine. Ophthalmologic examinations were performed weekly during the 10-week treatment period, every 2 weeks for an additional 6 weeks, and at 6 months after entry into the trial. RESULTS The median time to treatment failure (defined as worsening or no improvement of stromal keratitis or an adverse event) was 84 days (95% confidence interval, 69-93 days) for the acyclovir group and 62 days (95% confidence interval, 57-90 days) for the placebo group. By 16 weeks, 38 patients (75%) in the acyclovir group and 39 patients (74%) in the placebo group had failed treatment. Also by that time, the keratitis had resolved with trial medications, and there was no subsequent worsening in nine patients (18%) in the acyclovir group and ten (19%) in the placebo group. None of these results were significantly different between the two groups. However, visual acuity improved over 6 months in significantly more patients in the acyclovir group than in the placebo group. CONCLUSION There was no statistically or clinically significant beneficial effect of oral acyclovir in treating HSV stromal keratitis in patients receiving concomitant topical corticosteroids and trifluridine with regard to time to treatment failure, proportion of patients who failed treatment, proportion of patients whose keratitis resolved, time to resolution, or 6-month best-corrected visual acuity. Visual acuity improved over 6 months in more patients in the acyclovir group than in the placebo group.

Pathways of viral gene expression during acute neuronal infection with HSV-1

Pathways of viral gene expression were investigated during the acute phase of sensory ganglionic infection with HSV-1. To facilitate these studies we constructed KOS/62-3, an HSV-1 vector in which the Escherichia coli lac-Z gene was inserted behind both copies of the promoter for the viral latency-associated transcripts. Following footpad inoculation of mice with the virus, acutely infected dorsal root ganglion (DRG) neurons were assayed by dual immunofluorescence for the presence of beta-galactosidase and HSV viral antigens. Most infected neurons stained for either beta-galactosidase or viral antigens. Less than 0.2% of neurons staining for viral antigens also expressed beta-galactosidase, and less than 10% of neurons expressing beta-galactosidase also stained for viral antigen. As a consequence of these findings, we propose that there are essentially two populations of HSV-infected neurons during the acute phase of ganglionic infection. In one population of neurons there is abundant viral protein synthesis but minimal transcription of latency-associated transcripts, whereas in a second population of neurons viral gene expression is severely restricted except for the synthesis of latency-associated transcripts. Since DRG neurons are a heterogeneous population of cells, we further sought to determine whether either pathway of gene expression was more likely to occur in a particular neuronal phenotype. To accomplish this, antibodies were used to characterize the DRG neuronal phenotypes acutely infected with the virus. The results indicated that the pathway of neuronal infection characterized by transcription of abundant latency-associated transcripts and minimal viral protein synthesis was much more likely to occur in DRG neurons expressing the cellular antigen SSEA-3. These data indicate that the neuron plays a major role in regulating the outcome of infection with HSV. Finally, we sought to determine whether DNA replication occurs in the course of establishment of a latent infection. We found that the DNA content of neurons latently infected with KOS(M) strain HSV was not affected by treatment with nucleotide analogues during the acute phase of ganglionic infection, suggesting that viral DNA replication does not occur during the establishment of latent infection.

Varicella-Zoster Virus Retinitis in Patients With the Acquired Immunodeficiency Syndrome

We examined five patients infected with the human immunodeficiency virus who developed a rapidly progressive necrotizing retinitis characterized by early patchy choroidal and deep retinal lesions and late diffuse thickening of the retina. In all but one case, the retinitis began in the posterior pole with little or no clinical evidence of vasculitis. All five patients had relentless progression of disease and were left with atrophic and necrotic retinae, pale optic-nerve heads, and narrowed vasculature. None of the patients developed aqueous or vitreal inflammation or retinal detachment. Clinical and laboratory evidence suggested that varicella-zoster virus was the causal agent in all five cases. First, the onset of retinitis in four cases either succeeded or was coincident with an eruption of dermatomal zoster. Second, varicella-zoster virus was cultured from the two chorioretinal specimens and varicella-zoster virus antigen was detected in the vitreal aspirate from one case. Third, by means of immunocytochemistry, varicella-zoster virus antigen was found in the outer retinae of both enucleation specimens. Fourth, viral capsids with the size and shape of herpesviridae were found in the outer retinae of both enucleation specimens. The clinical features observed in this study are distinct from those described for the acute retinal necrosis syndrome and appear to constitute a new and highly characteristic pattern of varicella-zoster virus-induced disease.

A Sensitive and Specific Polymerase Chain Reaction-Based Assay for the Diagnosis of Cytomegalovirus Retinitis

PURPOSE To develop a sensitive and specific laboratory assay for the diagnosis of cytomegalovirus retinitis. METHOD We used a polymerase chain reaction-based assay for detection of cytomegalovirus DNA in vitreous samples. We attempted to detect cytomegalovirus DNA in 19 vitreous samples from patients with the acquired immunodeficiency syndrome (AIDS) who had untreated cytomegalovirus retinitis and in 40 vitreous samples from patients with AIDS who had been treated with systemic ganciclovir or foscarnet, or both. We also attempted to detect cytomegalovirus DNA in vitreous samples from 54 immunocompetent patients, including 32 with retinal detachment or macular hole, 11 with vitreous inflammation, and 11 with vitreous hemorrhage. Additionally, we attempted to detect cytomegalovirus DNA in 15 vitreous samples from patients with AIDS who had vitreoretinal inflammation not caused by cytomegalovirus. RESULTS Cytomegalovirus DNA was detected in 18 of 19 eyes with untreated cytomegalovirus retinitis. We detected cytomegalovirus DNA in 19 of 40 vitreous samples from patients with previously treated cytomegalovirus retinitis. Cytomegalovirus DNA was not detected in any of 69 patients who did not have a clinical diagnosis of cytomegalovirus retinitis. Thus, the assay had an estimated sensitivity of 95% in detecting untreated cytomegalovirus retinitis and a sensitivity of 48% in detecting cytomegalovirus retinitis that had been treated with systemic ganciclovir or foscarnet, or both. The assay did not give false-positive results in patients with vitreous hemorrhage or vitreous inflammation. Most important, the assay did not give false-positive results in AIDS patients with vitreous inflammation from causes other than cytomegalovirus retinitis. CONCLUSION We have developed a sensitive and specific diagnostic assay that will assist in the diagnosis of cytomegalovirus retinitis.

Herpes Simplex Virus Type 2 (HSV-2) Establishes Latent Infection in a Different Population of Ganglionic Neurons than HSV-1: Role of Latency-Associated Transcripts

ABSTRACT Herpes simplex virus type 1 (HSV-1) and HSV-2 cause very similar acute infections but differ in their abilities to reactivate from trigeminal and dorsal root ganglia. To investigate differences in patterns of viral infection, we colabeled murine sensory ganglia for evidence of HSV infection and for the sensory neuron marker A5 or KH10. During acute infection, 7 to 10% of HSV-1 or HSV-2 antigen-positive neurons were A5 positive and 13 to 16% were KH10 positive, suggesting that both viruses reach each type of neuron in a manner proportional to their representation in uninfected ganglia. In murine trigeminal ganglia harvested during HSV latency, 25% of HSV-1 latency-associated transcript (LAT)- and 4% of HSV-2 LAT-expressing neurons were A5 positive, while 12% of HSV-1 LAT- and 42% of HSV-2 LAT-expressing neurons were KH10 positive. A similar difference was observed in murine dorsal root ganglia. These differences could not be attributed to differences in LAT expression levels in A5- versus KH10-positive neurons. Thus, HSV-1 demonstrated a preference for the establishment of latency in A5-positive neurons, while HSV-2 demonstrated a preference for the establishment of latency in KH10-positive neurons. A chimeric HSV-2 mutant that expresses the HSV-1 LAT exhibited an HSV-1 phenotype, preferentially establishing latency in A5-positive neurons. These data imply that the HSV-1 and HSV-2 LAT regions influence the ability of virus to establish latency in different neuronal subtypes. That the same chimeric virus has a characteristic HSV-1 reactivation phenotype further suggests that LAT-influenced establishment of latency in specific neuronal subtypes could be an important part of the mechanism by which LAT influences viral reactivation phenotypes.