Polymerase chain reaction analysis of aqueous humor specimens in the diagnosis of cytomegalovirus retinitis in AIDS patients.

PURPOSE: To determine the value of the polymerase chain reaction analysis of aqueous humor specimens as a tool to diagnose cytomegalovirus retinitis in AIDS patients. METHODS: In all, 63 AIDS patients were evaluated in this study. They were sorted into two diagnostic categories: eyes with active cytomegalovirus retinitis and eyes without active cytomegalovirus retinitis. The aqueous humor and blood samples were collected and analyzed by polymerase chain reaction. RESULTS: A total of 49 patients had active cytomegalovirus retinitis (77.8%) and 14 patients had inactive cytomegalovirus retinitis or normal fundus (22.2%). The mean average of patients was 39 years (range: 22-59). The majority of patients were male (90.5%). Cytomegalovirus DNA was detected in 46 and 7 of 49 aqueous and blood samples, respectively, from AIDS patients with active cytomegalovirus retinitis. We did not detect cytomegalovirus DNA in any of the eyes without active cytomegalovirus retinitis. The sensitivity of polymerase chain reaction in the detection of cytomegalovirus in aqueous humor and blood samples was 93.5% and 14.3%, respectively. CONCLUSIONS: The polymerase chain reaction analysis is a safe, highly specific, and sensitive method to diagnose cytomegalovirus retinitis.

Relationship Between Opacity of Cytomegalovirus Retinitis Lesion Borders and Severity of Immunodeficiency Among People With AIDS.

Purpose: To evaluate risk factors for severity of cytomegalovirus (CMV) retinitis lesion whitening (opacity), using a standardized scoring system. Methods: We performed a cross-sectional, observational investigation of all individuals with newly diagnosed AIDS-related CMV retinitis in three randomized clinical trials and one prospective observational study. Opacity was scored by masked readers, using a prospectively defined ordinal 6-point scale. Demographic factors, laboratory data (CD4+, CD8+ T-lymphocyte counts, human immunodeficiency virus [HIV] blood levels), and lesion characteristics (location, size) were compared to the highest opacity score assigned to either eye. Among eyes with active lesions (scores ≥3), factors associated with severe opacity (scores 5, 6) were identified. Results: There were 299 participants (401 eyes with CMV retinitis). In one or more comparisons, increased opacity was associated with lower CD4+ and lower CD8+ T-lymphocyte counts, higher HIV blood level, lack of antiretroviral therapy, male sex, race/ethnicity, and bilateral disease. In eyes with active disease, severe opacity was associated with lower CD4+ T-lymphocyte count, higher HIV blood level, older age, Karnofsky score, lesion size, and bilateral disease. No relationship was identified between opacity and lesion location. Conclusions: Lesion border opacity (resulting from CMV activity) reflects level of immune function; as immunodeficiency becomes worse, CMV activity (and opacity) increases. The positive relationship between opacity and HIV blood level may reflect both immunodeficiency and increased CMV activity caused by transactivation of CMV by HIV. Scoring of opacity may be a useful, standard measure for continued study of CMV retinitis across different settings and populations. (Clinicaltrials.gov number for the HPMPC CMV Retinitis Trial: NCT00000142; Clinicaltrials.gov number for the Monoclonal Antibody CMV Retinitis Trial: NCT00000135; Clinicaltrials.gov number for the Ganciclovir-Cidofovir CMV Retinitis Trial: NCT0000014; Clinicaltrials.gov number for the Longitudinal Study of the Ocular Complications of AIDS: NCT00000168.).

Cytokine Profiles in Aqueous Humor and Plasma of HIV-infected Individuals with Ocular Syphilis or Cytomegalovirus Retinitis.

PURPOSE: To characterize the immunologic profile in aqueous humor (AqH) of HIV-infected individuals with cytomegalovirus retinitis (CMVr) or ocular syphilis and to assess if AqH and plasma represent independent cytokine compartments. METHODS: Concentrations of 27 cytokines in AqH and plasma of HIV-infected individuals with CMVr (n = 23) or ocular syphilis (n = 16) were measured by multiplex assay. Cytokine profiles of both groups were compared. RESULTS: Individuals with CMVr had higher plasma concentrations of interleukin (IL)-7, IL-8, IL-10, interferon (IFN)-γ, IFN-α2, G-CSF, IP-10 and IL-1α; as well as higher AqH concentrations of IL-1α, IP-10 and GM-CSF than those with ocular syphilis. AqH and plasma levels correlated only for IP-10 in both ocular infections. CONCLUSIONS: Individuals with CMVr had higher plasma cytokine levels than those with ocular syphilis. The immunologic profiles in AqH and plasma are independent. Therefore, AqH cytokine concentrations cannot be inferred from plasma cytokine concentrations in the population studied.

Long-term control of CMV retinitis in a patient with idiopathic CD4+ T lymphocytopenia.

Cytomegalovirus (CMV) retinitis with idiopathic CD4(+) T lymphocytopenia (ICL) is rare and difficult to control. We report a first case for long-term control of CMV retinitis with ICL using interleukin-2 (IL-2) therapy and succeeded in discontinuation of anti-CMV therapy. A 49-year-old Japanese woman was diagnosed with ICL based on low CD4(+) count (72/µl), negative for HIV-1 and -2 antibodies, and absence of any defined immunodeficiency diseases or immunosuppressive therapy. PCR test of the aqueous humor in the right eye was suggestive of CMV retinitis. She was treated with systemic ganciclovir, but after several relapses of CMV retinitis, rhegmatogenous retinal detachment appeared in the right eye and she became blind in that eye. Three years later, she developed CMV retinitis in the left eye. Although she received systemic and focal anti-CMV treatments, the retinitis showed no improvement. Finally, retinal detachment occurred, and she underwent vitrectomy. IL-2 was injected to increase CD4(+) counts. Because of hyperpyrexia, blepharedema, central scotoma, and color anomaly, we changed to low-dose IL-2 therapy with no side effects. Finally, we succeeded in increasing the CD4(+) count to more than 200/µl after discontinuation of low-dose IL-2 therapy. CMV retinitis never recurred after discontinuation of anti-CMV therapy, with good visual acuity of 20/20 in the left eye. She developed blindness of the first affected right eye, whereas the visual acuity of the left eye remains excellent more than 12 years after the onset of CMV retinitis through the combined use of anti-CMV therapy, IL-2 therapy, and vitrectomy.

Hemorheologic abnormalities associated with HIV infection: in vivo assessment of retinal microvascular blood flow.

PURPOSE: To evaluate retinal microvascular blood flow in human immunodeficiency virus (HIV)-infected individuals using scanning laser Doppler flowmetry (SLDF) and to seek correlations between flow and various laboratory measures that may predict alterations in flow. METHODS: The Heidelberg Retina Flowmeter and SLDF software were used to acquire in vivo retinal blood flow data from 24 HIV-infected individuals and 16 HIV-negative control subjects. In each subject, separate scans were performed in each of six retinal regions: nasal parapapillary retina; macula; and the superior, nasal, inferior, and temporal periphery. Erythrocyte aggregation (assessed in vitro by a fully automatic erythrocyte aggregometer and by zeta sedimentation ratio [ZSR, a hematocrit-independent sedimentation rate]), serum fibrinogen level, plasma viscosity, and leukocyte rigidity (assessed in vitro with a cell transit analyzer) were compared with flow in selected regions. RESULTS: Flow was significantly higher in the periphery (superior, nasal, inferior, temporal) than in the posterior retina (nasal parapapillary retina, macula). Flow was highest in the temporal periphery for both HIV-infected subjects and control subjects. Flow in the posterior retina was significantly lower in HIV-infected subjects than in control subjects (P < 0.0001). Among HIV-infected individuals, flow in the macula correlated negatively with ZSR (r = -0.397, P = 0.0547) and leukocyte rigidity (r = -0.505, P = 0.0119). CONCLUSIONS: Microvascular blood flow in the posterior retina is reduced in HIV-infected individuals. Both increased erythrocyte aggregation and increased leukocyte rigidity contribute to this hemorheologic abnormality.

Cytokine profile in response to Cytomegalovirus associated with immune recovery syndrome after highly active antiretroviral therapy.

BACKGROUND: Several changes have occurred in the presentation and course of cytomegalovirus (CMV) retinitis in patients with AIDS since the introduction of HAART (highly active antiretroviral therapy). In some individuals who take HAART, retinitis is kept under control even after the discontinuation of anti-CMV therapy. However, many of these patients develop intraocular inflammation. Uveitis, cataract, vitreitis, cystoid macular edema, epiretinal membrane, and disc edema may occur in patients with immune recovery syndrome (IRS). METHODS: We evaluated the CMV-specific immune response in 55 patients by assessing CMV-specific lymphocyte proliferation, cytotoxicity, and cytokine production and correlated it with the clinical outcome. RESULTS: Our data suggest that control of CMV retinitis is associated with acquisition of cytotoxic and lymphoproliferative responses to CMV. In addition, the upsurge of macular and disc edema seems associated with the production of interleukin-4 and tumor necrosis factor-alpha, whereas vitreitis is associated with the production of interleukin-2 and interferon-gamma. INTERPRETATION: The type of T-cell response that develops after HAART may determine the side effects of immune recovery and these effects are predictable based on the lymphokine profile produced by CMV-specific cells.

Influence of filgrastim (granulocyte colony-stimulating factor) on human immunodeficiency virus type 1 RNA in patients with cytomegalovirus retinitis.

Filgrastim, or granulocyte colony-stimulating factor, reverses neutropenia associated with human immunodeficiency virus type 1 (HIV-1) and cytomegalovirus (CMV) infections. During a trial of anti-CMV retinitis therapies coadministered with antiretroviral therapy, 2-4 plasma specimens of HIV-1 RNA were collected from 36 HIV-1-infected patients receiving filgrastim to prevent neutropenia and from 36 patients not receiving filgrastim. For both groups, the crude mean and mean rate of change of HIV-1 log(10) RNA levels were similar. Adjustment for covariates (CD4(+) T cell lymphocytes, virus load at enrollment, level of neutropenia and antiretroviral therapy [mainly non-highly active antiretroviral therapy], and anti-CMV therapy during follow-up) resulted in a mean log(10) HIV-1 RNA level for individuals receiving filgrastim versus those not receiving the drug of 5.11 versus 4.87 (P=.12) and respective log mean rates of change per month of -0.08 versus -0.21 (P=.08). This latter difference has borderline statistical significance, which suggests that filgrastim may reduce the decline of HIV-1 RNA loads.

An evaluation of serum soluble CD30 levels and serum CD26 (DPPIV) enzyme activity as markers of type 2 and type 1 cytokines in HIV patients receiving highly active antiretroviral therapy.

This study evaluates serum CD26 (dipeptidyl peptidase IV, DPPIV) enzyme activity and serum levels of soluble CD30 as markers of T1 and T2 cytokine environments in HIV patients who achieved immune reconstitution after highly active antiretroviral therapy (HAART). Patients who had experienced inflammatory disease associated with pre-existent opportunistic infections after HAART (immune restoration diseases, IRD) were considered separately. Serum sCD30 levels and CD26 (DPPIV) enzyme activity were compared with IFN-gamma production by PBMC cultured with cytomegalovirus (CMV) antigen in controls and patient groups. High sCD30 levels were associated with low IFN-gamma production after antigenic stimulation in control subjects and, to a lesser extent, in immune reconstituted HIV patients. There was no association between serum CD26 (DPPIV) enzyme activity and IFN-gamma production or sCD30 levels. Serum sCD30 levels and CD26 (DPPIV) enzyme activity were significantly increased in immune reconstituted patients with high HIV viral loads. Patients who had experienced CMV retinitis as an IRD had significantly higher sCD30 levels than all other patient groups. Hence, high sCD30 levels may be a marker of a T2 cytokine environment in HIV patients with immune reconstitution and are associated with higher HIV viral loads and a history of CMV associated IRD.

[Anterior uveitis and cidofovir]. / Uvéite antérieure et cidofovir.

PURPOSE: This retrospective study was designed to determine the different parameters involved in the occurrence of uveitis during treatment with codofovir. PATIENTS AND METHODS: This study included 10 patients out of 13 treated with cidofovir for cytomegalovirus (CMV) disease. Ocular examination, CD4+ lymphocyte count, and creatinine clearance were performed for each case of uveitis. RESULTS: During the 17-month study, 20 uveitis cases were analyzed. The first attack occurred after a median interval of 7.6 doses. At the time of ocular inflammation, 65% of the cases had a CD4+ lymphocyte count >=100x10(6)/L, the patients thus had an improved immune function. Half of the patients had a normal creatinine clearance. The patients with a CD4+ lymphocyte count<100x10(6)/L who presented one or more incidents of uveitis had an abnormal clearance, thus probably inducing intraocular storage of the drug. CONCLUSION: The occurrence of anterior uveitis during treatment with cidofovir is induced by the association of several parameters: a previous history of CMV retinitis, improvement of the immune function state, and intraocular storage of the drug.

Quantitative CMV antigenemia correlated with ophthalmoscopic screening for CMV retinitis in AIDS patients.

To assess the efficacy of the cytomegalovirus (CMV) antigen test in detecting the clinical presence of CMV retinitis. A retrospective chart review was conducted on 86 HIV positive patients who underwent dilated fundus exams for CMV retinitis. All patients had a CMV antigen assay performed within three months of their retinal exam. At a level of 45, the antigen test has a sensitivity of 96% in correctly detecting CMV retinitis and a specificity of 90.2%. The negative and positive predictive values of the antigen test were 98.2% and 80%, respectively. CMV antigen blood test provides a useful screening tool in detecting the presence or absence of CMV retinitis. An antigen level less than 45 strongly suggests the absence of retinitis.

Monitoring plasma levels of ganciclovir in AIDS patients receiving oral ganciclovir as maintenance therapy for CMV retinitis.

OBJECTIVES: To investigate whether low ganciclovir serum levels in patients on maintenance oral ganciclovir therapy are associated with recurrence of CMV retinitis. METHODS: A prospective study of the plasma concentration of ganciclovir after initiation of maintenance oral ganciclovir therapy in 14 AIDS patients who had recovered from acute cytomegalovirus (CMV) retinitis. RESULTS: Five of the 14 patients exhibited a mean time to recurrence of 37 days. The mean trough plasma concentration of ganciclovir in these patients after 1 month of oral ganciclovir therapy, was 0.40 +/- 0.30 mg/L. Nine patients had a mean time of progression of 263 days. The mean trough plasma concentration of ganciclovir in the latter patients was 0.80 +/- 0.60 mg/L. CONCLUSIONS: Patients exhibiting trough plasma levels of ganciclovir below 0.6 mg/L may be at higher risk of progression than patients who exhibited levels above 0.6 mg/L.

Quantitation of peripheral blood cytomegalovirus DNA for monitoring recurrent cytomegalovirus retinitis in pediatric solid organ transplant recipients.

Cytomegalovirus (CMV) infection is a major concern following solid organ transplantation, especially in the pediatric population who remain at high risk of primary infection. CMV disease leads not only to increased patient and graft morbidity, but also to increased health care costs. This study describes the usefulness of a quantitative CMV polymerase chain reaction (PCR) technique for monitoring peripheral blood CMV DNA in pediatric recipients of kidney and liver allografts who had recurrent CMV retinitis. The incidence of CMV disease in 28 pediatric transplant recipients was 28.6%, one-half of whom developed retinitis. Two of these patients had recurrent retinitis on cessation of anti-viral treatment. A peripheral blood CMV DNA copy number of > or =500/microg of DNA was associated with recrudescence of the retinitis in these patients. We conclude that the measurement of peripheral blood CMV DNA by PCR is a useful tool for the surveillance of disease resolution and recurrence. This is particularly important in patients with CMV retinitis, who may remain asymptomatic for a period of time, despite recurrences.

Role of the cytomegalovirus (CMV)-antigenemia assay as a predictive and follow-up detection tool for CMV disease in AIDS patients.

Forty-two patients were evaluated to determine the value of the CMV antigenemia (CMV-Ag) test as a follow-up marker as well as a prediction marker of CMV disease. Twenty patients were positive for at least one positive CMV-Ag assay and 9 of them developed CMV retinitis. With the threshold value (10 positive cells), sensitivity was 56% and specificity was 94%. The CMV-Ag assay, with the threshold value, produced high specificity, positive predictive value and negative predictive value but relatively poor sensitivity. Eight patients experienced CMV disease relapse a total of 16 times. At relapse, 8 of the 16 times showed negative for CMV-Ag assay; 7 underwent systemic maintenance while 1 underwent local maintenance. It is inferred that the CMV-Ag test is a poor follow-up marker to detect the relapse of CMV disease, particularly in patients undergoing systemic maintenance.

Human cytomegalovirus glycoprotein B genotypes in blood of AIDS patients: lack of association with either the viral DNA load in leukocytes or presence of retinitis.

It has been suggested that human cytomegalovirus (HCMV) glycoprotein B (gB) genotypes could be used as a marker for viral virulence in patients with AIDS. The present study was designed to evaluate a possible association between specific gB genotypes, the presence of HCMV retinitis, and the HCMV viral load. Fifty-four blood samples were obtained from 54 HIV- and HCMV-infected patients. Twenty-seven of these patients were asymptomatic for HCMV, whereas the other 27 patients had been diagnosed recently with HCMV retinitis. HCMV gB genotyping was carried out by using restriction enzyme analysis of PCR-amplified PMNL extracts. Determination of the HCMV viral load in the same specimens was carried out using a quantitative-PCR. HCMV gB genotype 2 was found more frequently than other genotypes in PCR-amplified polymorphonuclear leukocytes (PMNL) of patients with AIDS (P < 0.05) but not more frequently in samples from patients with HCMV retinitis. No significant association was found between any HCMV gB genotypes and the viral load in blood. In conclusion, the actual HCMV gB genotyping system using PMNL provides no additional benefit over the viral load in blood for identification of HIV-infected subjects at risk of HCMV disease.

Cytomegalovirus (CMV) retinitis activity is accurately reflected by the presence and level of CMV DNA in aqueous humor and vitreous.

To evaluate the potential of ocular and systemic specimens to provide markers of active cytomegalovirus (CMV) retinitis, we examined the relationship between virologic and clinical aspects of CMV infections in AIDS patients with CMV retinitis. CMV polymerase chain reaction (PCR) analysis of 74 aqueous humor and vitreous specimens indicated that ocular specimens can provide accurate markers to differentiate active and inactive CMV retinitis (aqueous or vitreous PCR, P<.001). Moreover, these markers were superior to extraocular measures, including plasma PCR (P=.08) and blood and urine CMV cultures (P=.05). A direct correlation was identified between the quantity of CMV DNA in aqueous humor or vitreous specimens and the corresponding surface area of active CMV retinitis (r2=.69 and.44, respectively). Thus, qualitative and quantitative PCR-based analyses of aqueous humor can provide valuable markers of CMV retinitis activity. Such assays could provide rapid and reliable tools to assist in management of patients with CMV retinitis in whom the view of the retina is obscured.

CMV retinitis after cessation of ganciclovir therapy for CMV antigenemia in an unrelated BMT recipient.

An 11-year-old boy with severe aplastic anemia underwent unrelated BMT following TBI, antithymocyte globulin and CY. On day +23, CMV antigenemia was detected which resolved with ganciclovir. Eight days after discontinuing ganciclovir, he complained of impaired visual acuity. Ophthalmologic findings and a positive PCR study using anterior chamber fluid from the right eye confirmed the presumptive diagnosis of CMV retinitis, although CMV antigenemia and PCR studies using PBMC were then negative. He was successfully re-treated with ganciclovir. CMV retinitis should be considered even when CMV antigenemia is not present or PCR using PBMC is negative.

Cytomegalovirus (CMV) strain differences between the eye and blood in AIDS patients with CMV retinitis.

OBJECTIVE: To investigate possible differences in cytomegalovirus (CMV) strain distribution between the eye and blood in AIDS patients with CMV retinitis. METHODS: CMV DNA sequences from aqueous humour and peripheral blood leukocytes (PBL), obtained from 13 AIDS patients with CMV retinitis, were compared. DNA was isolated and the CMV IE-1 sequence (part of the immediate early-1 gene) and the a-sequence (located in the a-region) were amplified by polymerase chain reaction (PCR). The PCR products of the a-sequence were analysed by Southern blotting for amplified fragment-length polymorphisms. The level of divergence between the a-sequences of aqueous humour- and PBL-derived CMV was studied in two patients by cloning these sequences followed by sequence analysis. RESULTS: CMV DNA could be detected in all aqueous humour samples and in 10 out of 13 paired blood samples. In the 10 patients, with CMV DNA detectable in both aqueous humour and PBL, seven cases showed differences between the amplified products of both compartments. Sequence analysis in two patients revealed that the aqueous humour and PBL of the same patient can harbour both identical, similar and highly divergent CMV a-sequences. CONCLUSION: These results indicate that despite the haematogenous spread of CMV, the eye, being a relatively shielded organ, may contain CMV strains different from those found in the blood.

Cytomegalovirus glycoprotein B genotyping in ocular fluids and blood of AIDS patients with cytomegalovirus retinitis.

PURPOSE: To determine the frequency of cytomegalovirus glycoprotein B (gB) genotypes in clinical samples of ocular fluids of patients with acquired immune deficiency syndrome (AIDS) who have cytomegalovirus retinitis and to compare these with the cytomegalovirus gB genotype in paired peripheral blood leukocytes. METHODS: Glycoprotein B genotypes of cytomegalovirus genomic DNA were determined in 29 ocular and 9 paired blood samples of 27 patients, by polymerase chain reaction amplification followed by restriction fragment length polymorphism analysis. RESULTS: In the 29 ocular samples, 30 gB genotypes were determined: Glycoprotein B1 was found in 8 samples (27%), gB2 in 9 samples (30%), gB3 in 6 samples (20%), and gB4 in 3 samples (10%). In one sample, a mixed genotype was observed. In addition to these previously characterized gB genotypes, a new gB variant was observed in the ocular fluid of four patients. Partial sequence analysis revealed that this new gB genotype is closely related to gB3, and it was therefore named gB3'. In the blood samples, only gB1, gB2, and gB3 genotypes were observed. In the nine paired samples of ocular fluid and blood, four showed a difference in gB genotype between these compartments. CONCLUSIONS: The distribution of cytomegalovirus glycoprotein B genotypes gB1-gB4 in ocular fluids of patients with AIDS who have cytomegalovirus retinitis was determined in this study. The predominance of gB2, as described by others, was not confirmed. The glycoprotein B genotype in the eye can be different from the genotype found in the blood of the same patient. A new gB variant, gB3', was found in the ocular samples of 4 of 27 patients, but not in the blood samples tested.