Detecting herpesvirus DNA in uveitis using the polymerase chain reaction.

BACKGROUND: Herpesviruses are involved in the pathogenesis of many ocular diseases including keratitis, iridocyclitis, and acute retinal necrosis syndrome. The rapid and accurate diagnosis of herpetic infections has become increasingly important with the rising incidence of immunosuppressive diseases. The purpose of this study was to evaluate the use of the polymerase chain reaction (PCR) to detect herpesvirus DNA in uveitis patients. METHODS: Aqueous samples were aspirated from 11 patients with active uveitis of suspected viral origin. Using PCR, masked samples were assayed for herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV) to assist in supporting the clinical diagnosis of viral aetiology. Masked controls included 10 aqueous humour specimens from normal patients undergoing cataract surgery and specimens from seven patients diagnosed with active non-viral uveitis--Behçet's disease, sarcoidosis, Fuchs' heterochromic iridocyclitis, or Harada's disease. RESULTS: Ten of 11 cases clinically diagnosed as being of possible viral aetiology yielded aqueous PCR positive for a herpesvirus. Eight patients were PCR positive for amplified HSV DNA, of whom two had acute retinal necrosis, one had corneal endotheliitis, and five had recurrent iridocyclitis. VZV DNA was detected in one case of iridocyclitis, and CMV DNA in one case of chorioretinitis. Successful therapy was based on the PCR results. Ten normal aqueous specimens and the seven uveitis samples from cases not suspected of a viral aetiology were PCR negative for HSV, VZV, and CMV. CONCLUSION: These results demonstrate that detecting herpesvirus DNA in the aqueous humour is useful to support a clinical diagnosis of viral uveitis.     

Detection of herpesvirus genome by multiplex polymerase chain reaction (PCR) and real-time PCR in ocular fluids of patients with acute retinal necrosis

PURPOSE: The human herpesvirus (HHV) family consists of types 1 to 8 (HHV1-8). The purpose of this study was to investigate the detection of HHV DNA, especially HSV1 (herpes simplex virus 1, HHV1), HSV2 (herpes simplex virus 2, HHV2), and VZV (varicella-zoster virus, HHV3) in ocular fluids of patients with acute retinal necrosis(ARN). METHODS: The intraocular genome for HHV1-8 was determined in 19 ocular fluid samples (12 vitreous fluid and 7 aqueous humor samples) taken from ARN patients (n=14). The samples were tested for the presence of virus DNA by two systems of polymerase chain reaction (PCR): the multiplex PCR screening test and real-time quantitative PCR. RESULTS: Multiplex PCR demonstrated VZV (n=16, 84%), HSV1 (n = 1.5%) or HSV2 (n = 2.11%)genomic DNA in all the samples. In real-time PCR, a high copy number of virus DNA was detected. The virus DNA-positive samples contained Epstein-Barr virus (EBV, HHV4) DNA in 9 of 19 samples (47%). No HHV6-8 DNA was detected in the ocular samples, and no virus DNA was detected in the serum samples. CONCLUSIONS: The genome for HHV1-3 was detected in the patients with ARN. All cases contained a high copy number for the virus DNA that indicates viral replication. PCR systems are useful for determing whether virus infections are associated with uveitis.     

Multiplex polymerase chain reaction for detection of herpes simplex virus type 1, type 2, cytomegalovirus,and varicella-zoster virus in ocular viral infections

PURPOSE: To detect simultaneously herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), cytomegalovirus (CMV), and varicella-zoster virus (VZV) in ocular specimens suspected of indicating viral infection, and to compare the results of multiplex polymerase chain reaction (PCR) with those of uniplex PCR. METHODS: Forty specimens, collected from 33 patients with clinically suspected herpes virus ocular infection, were tested. DNA was extracted from the specimens and amplified by multiplex and uniplex PCR. RESULTS: Both multiplex PCR and uniplex PCR gave the same results. Nineteen (19/33, 57.6%) patients were PCR-positive, among whom HSV-1 was detected in 13 (13/19, 68.4%) patients, and VZV in 6 (6/19, 31.6%) patients. CONCLUSION: These results demonstrated that multiplex PCR is as reliable as uniplex PCR, and is an accurate and a cost-saving method to identify several agents from a single specimen.     

Rose bengal and lissamine green inhibit detection of herpes simplex virus by PCR

PURPOSE: To determine whether rose bengal and lissamine green affect polymerase chain reaction (PCR) detection of herpes simplex virus (HSV). DESIGN: Laboratory investigation. METHODS: Diagnostic corneal scrapings were evaluated for PCR inhibitory activity. Dacron swabs inoculated with rose bengal and lissamine green were processed as clinical samples, inoculated with control HSV, varicella zoster (VZV), cytomegalovirus (CMV), and toxoplasma DNA and prepared for PCR. The effects of calcium alginate and cotton swabs were also evaluated. RESULTS: Rose bengal, lissamine green, and calcium alginate not only inhibit PCR detection of HSV DNA, but also detection of VZV, CMV, and toxoplasma DNA. This inhibition could be overcome by serial dilution and by DNA purification of the sample before PCR. CONCLUSIONS: Rose bengal, lissamine green, and calcium alginate can inhibit PCR detection of HSV DNA. Clinical scrapings to be sent for PCR diagnostic testing should be taken before instillation of rose bengal or lissamine green.     

Cytomegalovirus in aqueous humor from an eye with corneal endotheliitis

PURPOSE: To report cytomegalovirus (CMV) DNA in aqueous humor from a patient with unilateral corneal endotheliitis. DESIGN: Case report. METHODS: A 51-year-old man presented with unilateral corneal endotheliitis with linear keratic precipitates and coin-shaped lesions. Tear and aqueous humor samples were subjected to polymerase chain reaction to look for DNA from herpes simplex virus (HSV), varicella zoster virus (VZV), and CMV. RESULTS: Aqueous humor from the diseased eye contained DNA from CMV but not HSV or VZV. Its specificity was confirmed by Southern blot tests. Intravenous ganciclovir treatment resulted in the localization of his corneal edema and the reduction in keratic precipitates. There was severe destruction of corneal endothelial cells. CMV DNA was not detected in tears or control samples. CONCLUSIONS: In this healthy man with corneal endotheliitis, we detected CMV DNA in aqueous humor from the affected eye, but not HSV or VZV. This suggests that CMV may cause corneal endotheliitis in patients without immunodeficiency.     

The diagnostic significance of enzyme linked immuno-sorbent assay for herpes simplex,varicella zoster and cytomegalovirus retinitis

PURPOSE: To evaluate the diagnostic usefulness of enzyme linked immuno-sorbent assay (ELISA) in single serum samples to associate herpes simplex virus (HSV), varicella zoster virus (VZV) or cytomegalovirus (CMV) with viral retinitis as against polymerase chain reaction (PCR) on intraocular specimens. It was also designed to study the seroprevalence in normal healthy individuals, and the genomic prevalence of HSV, VZV and CMV in patients without an active viral inflammatory process. METHODS: PCR for the detection of HSV, VZV and CMV genomes was done on 33 and 90 intraocular fluids from viral retinal patients and non-viral controls respectively. ELISA was done on 30 and 100 serum samples from viral retinitis patients and normal healthy controls respectively. RESULTS: PCR did not detect HSV, VZV and CMV genomes except one, in which VZV-DNA was detected. ELISA showed prevalence rates of 28%, 83% and 90% for antibodies against HSV, VZV and CMV respectively in the normal population. In the 30 viral retinitis patients, PCR detected HSV-DNA in 2 (6.7%), VZV-DNA in 7 (23.3%) and CMV-DNA in 6 (20.0%) patients, while ELISA detected antibodies against HSV, VZV and CMV in 13 (43.3%), 24 (80.0%) and 23 (76.7%) patients respectively. ELISA was of value in indirect diagnosis only in 6 (20.0%) as compared to 15 (50.0%) of 30 patients by PCR, this difference was statistically significant (McNemar test, P value = 0.005). CONCLUSION: Serology by ELISA is no longer a useful diagnostic tool to associate HSV, VZV and CMV viruses with viral retinitis.     

Detection of herpes simplex virus type 1, 2 and varicella zoster virus DNA in recipient corneal buttons

AIM: To study the value of polymerase chain reaction (PCR) analysis, to detect viral DNA in recipient corneal buttons taken at the time of penetrating keratoplasty (PKP) in patients with an initial diagnosis of herpetic stromal keratitis (HSK). Since HSK has a tendency to recur, an accurate diagnosis of previous HSK could be the reason to start antiviral treatment immediately, thereby possibly decreasing the number of graft failures due to recurrent herpetic keratitis. METHODS: Recipient corneal buttons and aqueous humour (AH) samples were obtained at the time of PKP from HSK patients (n=31) and from other patients (n=78). Eye bank corneas were also used (n=23). Herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), and varicella zoster virus (VZV) infection were assessed by PCR and antibody detection. RESULTS: The clinical diagnosis HSK could be confirmed by PCR for HSV-1 in 10/31 (32%). In these corneal buttons HSV-2 DNA was detected in 1/31 (3%) and VZV DNA in 6/31 (19%). Intraocular anti-HSV antibody production was detected in 9/28 AH samples tested (32%). In the other patient derived corneas HSV-1 DNA was detected in 13/78 (17%), including eight failed corneal grafts without clinically obvious herpetic keratitis in the medical history. In clear eye bank corneas HSV-1 was detected in 1/23 (4%). CONCLUSIONS: PCR of HSV-1 on corneal buttons can be a useful diagnostic tool in addition to detection of intraocular anti-HSV antibody production. Furthermore, the results were suggestive for the involvement of corneal HSV infection during allograft failure of corneas without previous clinical characteristic signs of herpetic keratitis.     

The prevalence of herpes family virus DNA in the conjunctiva of patients positive and negative for human immunodeficiency virus using the polymerase chain reaction

ObjectiveTo help understand the pathogenesis of herpes family virus ocular infection among patients positive for HIV, the authors compared the rates of detection of herpes family virus DNA from the conjunctiva of patients who are positive and negative for human immunodeficiency virus (HIV) using the polymerase chain reaction (PCR).DesignCross-sectional study.ParticipantsThe conjunctival scrapings of 30 patients positive for HIV and 30 patients negative for HIV were examined.InterventionPCR was used to assay for the presence of herpes simplex virus type 1 (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) DNA (n = 240 samples).Main outcome measureThe rate of detection of virus DNA in the two groups, controlling for age, gender, and race, was measured.ResultsHSV and VZV DNA were not detected in any of the HIV-positive or HIV-negative samples. CMV DNA was detected in 20% (6 of 30) of patients positive for HIV and was undetected in control subjects negative for HIV (P = 0.01). EBV DNA was detected in 40% (12 of 30) of patients positive for HIV and in 47% (14 of 30) of control subjects negative for HIV (P = 0.58).ConclusionsThere was no difference in the frequency of detection of HSV, VZV, or EBV DNA from the conjunctiva of patients positive or negative for HIV. Only CMV DNA was detected at a significantly higher rate in the conjunctiva of patients positive for HIV compared with control subjects negative for HIV. These different rates of peripheral virus shedding may be one possible explanation for the different rates of clinical infection among the herpes family viruses among patients positive for HIV.     

Rubella virus is associated with fuchs heterochromic iridocyclitis

PURPOSE: To determine whether rubella virus (RV) is involved in the pathogenesis of Fuchs heterochromic iridocyclitis (FHI). DESIGN: Retrospective patient-controlled study. METHODS: Intraocular immunoglobulin G production against RV, herpes simplex virus (HSV), varicella zoster virus (VZV), and Toxoplasma gondii was determined in the aqueous humor of 14 patients with FHI, 13 control subjects with herpetic uveitis anterior, and 19 control subjects with ocular toxoplasmosis by calculation of the Goldmann-Witmer coefficient (GWC). RESULTS: All patients and control subjects were seropositive for RV. Intraocular antibody production (GWC >3) against RV was found in 13 of 14 patients (93%) with FHI. Intraocular antibody production against HSV, VZV, or T gondii was not detected. None of the control subjects with herpetic uveitis anterior or with toxoplasma chorioretinitis had a positive GWC for rubella virus (P < .0001, Fisher exact test). CONCLUSION: Rubella virus, but not HSV, VZV, or T gondii, is associated with FHI.     

Polymerase chain reaction for diagnosis of herpetic intraocular inflammation

The authors present a polymerase chain reaction method for rapid and direct diagnosis of herpetic intraocular infections using small volume samples of intraocular fluid from 29 patients with various intraocular inflammatory diseases and 24 controls with senile cataract. Of six patients with early acute retinal necrosis from whom aqueous humor was tested, four were found to be positive for the presence of varicella-zoster (VZV) DNA while the other two were positive for the presence of herpes simplex virus (HSV) DNA. One of the patients with HSV DNA had been tested at an extremely early stage, at which time the aqueous humor viral antibody ratio did not predict a specific viral infection. Among four patients with acute retinal necrosis in relatively late stages following treatment with acyclovir from whom vitreous was obtained and tested, only one was found to have the presence of any viral DNA (VZV). On the other hand, the vitreous viral antibody ratio was found to be predictive of VZV infection in all four cases. VZV DNA was also detected in aqueous humor samples from four patients with suspected herpes zoster anterior uveitis, while HSV DNA was found in the aqueous humor of one patient with nonspecific keratouveitis. Neither human cytomegalovirus DNA nor human herpesvirus-6 DNA was detected in any sample included in this study. Finally, Epstein-Barr virus DNA was detected in the aqueous humor of the majority of patients studied and identified in cataract patients as well, suggesting either low specificity of the authors' assay for this virus or ubiquity of this virus in human eyes. In summary, the PCR method proved to be a very useful tool in establishing an etiological diagnosis in patients in the early stages of acute retinal necrosis, and in patients with anterior uveitis due to suspected HSV or VZV infection.     

Polymerase chain reaction for diagnosis of herpetic intraocular inflammation

The authors present a polymerase chain reaction method for rapid and direct diagnosis of herpetic intraocular infections using small volume samples of intraocular fluid from 29 patients with various intraocular inflammatory diseases and 24 controls with senile cataract. Of six patients with early acute retinal necrosis from whom aqueous humor was tested, four were found to be positive for the presence of varicella-zoster (VZV) DNA while the other two were positive for the presence of herpes simplex virus (HSV) DNA. One of the patients with HSV DNA had been tested at an extremely early stage, at which time the aqueous humor viral antibody ratio did not predict a specific viral infection. Among four patients with acute retinal necrosis in relatively late stages following treatment with acyclovir from whom vitreous was obtained and tested, only one was found to have the presence of any viral DNA (VZV). On the other hand, the vitreous viral antibody ratio was found to be predictive of VZV infection in all four cases. VZV DNA was also detected in aqueous humor samples from four patients with suspected herpes zoster anterior uveitis, while HSV DNA was found in the aqueous humor of one patient with nonspecific keratouveitis. Neither human cytomegalovirus DNA nor human herpesvirus-6 DNA was detected in any sample included in this study. Finally, Epstein-Barr virus DNA was detected in the aqueous humor of the majority of patients studied and identified in cataract patients as well, suggesting either low specificity of the authors' assay for this virus or ubiquity of this virus in human eyes. In summary, the PCR method proved to be a very useful tool in establishing an etiological diagnosis in patients in the early stages of acute retinal necrosis, and in patients with anterior uveitis due to suspected HSV or VZV infection.     

DNA of cytomegalovirus detected by PCR in aqueous of patient with corneal endotheliitis after penetrating keratoplasty

PURPOSE: Corneal endotheliitis often leads to severe endothelial dysfunction and can be caused by herpes simplex virus (HSV), varicella zoster virus (VZV), and other viruses (eg, the mumps virus). We report a case of corneal endotheliitis caused by cytomegalovirus (CMV) that developed after a penetrating keratoplasty. METHODS: A complete ophthalmologic examination was performed on a patient with corneal endotheliitis that developed after a penetrating keratoplasty. To determine the cause of the endotheliitis, polymerase chain reaction (PCR) was used to amplify the DNA of HSV, VZV, and CMV in samples of the aqueous humor. RESULTS: Slit-lamp biomicroscopy showed a moderate stromal edema in the upper temporal part of the transplanted cornea along with keratic precipitates (KPs) arranged in a coin-shaped pattern. Repeated treatments with steroids and acyclovir were only temporarily successful. PCR detected the DNA of CMV in an aqueous sample, and the treatment was switched to topical and systemic application of ganciclovir. This resulted in the disappearance of the KPs and resolution of the stromal edema within 2 weeks. CONCLUSIONS: From the PCR results and the favorable response to ganciclovir, the corneal endotheliitis was most likely caused by cytomegalovirus in this case.     

Effects of topical anaesthetics and fluorescein on the real-time PCR used for the diagnosis of Herpesviruses and Acanthamoeba keratitis

BACKGROUND: The early microbiological diagnosis of corneal infections may prevent the condition from worsening. AIM: To study the potential interferences of oxybuprocain and fluorescein solutions used by ophthalmologists on the performances of the real-time polymerase chain reaction (PCR) carried out as routine test for diagnosis of keratitis. METHODS: Quantified suspensions of Herpes simplex virus (HSV1), Varicella zoster virus (VZV), Cytomegalovirus (CMV) and Acanthamoeba with and without oxybuprocain or fluorescein added before DNA extraction were tested by real-time PCR. RESULTS: The capacities of the real-time PCR to detect HSV, VZV, CMV and Acanthamoeba were reduced by oxybuprocain and fluorescein. Both products diluted to 1/16 reduced the PCR detection capacities for more than 2 logs (DNA copies/sample). CONCLUSIONS: The simultaneous introduction of fluorescein or topical anaesthetics into the tubes containing the specimens to be tested by PCR may lead to false negative results. Because corneal specimens for microbiological diagnosis of keratitis are obtained after topical administration of anaesthetics and corneal staining with fluorescein, ophthalmologists should be aware to rinse the eye surface intensively with appropriate eye solutions to minimise the risks of misdiagnosis.     

Corneal buttons obtained from patients with HSK harbor high copy numbers of the HSV genome

PURPOSE: To detect herpes simplex virus (HSV) genome in the cornea, we sampled the limbal corneas and scleras of the imported eye bank eyes and recipient's corneal buttons and quantitated HSV genome in them by real-time polymerase chain reaction (PCR). METHODS: Forty-four recipient corneas including 7 corneas with and 37 corneas without a history of herpetic keratitis, 70 eye bank donor limbal corneas, and 35 eye bank donor scleras were obtained. Primers for real-time PCR were synthesized using the HSV-1 and -2 common regions of the viral DNA polymerase. Primers for conventional PCR were designed to detect HSV-1 and -2 and varicella zoster virus (VZV). RESULTS: Significantly higher copy number of HSV DNA was detected in corneas with a history of herpetic keratitis 85.7% (6/7), with an average of 1.6 x 10(4) copies/mg tissue weight than in corneas without a history of herpetic keratitis 10.8% (4/37), with an average of 8.7 copies/mg tissue weight (P < 0.05, Mann-Whitney U test). HSV DNA was detected in 5.7% (4/70) of the eye bank donor corneas, with an average of 4.9 x 10(2) copies/mg tissue weight, and in 8.6% (3/35) of the donor scleras, with an average of 10.6 copies/mg tissue weight. HSV-2 and VZV-DNA were not detected in these samples. CONCLUSIONS: Real-time PCR quantitated HSV genome in the cornea even at a quiescent phase of infection. HSV genome was detected in the corneas and scleras without a past history of herpetic keratitis by this method.     

Association of herpesviruses in the aqueous humor of patients with serpiginous choroiditis: a polymerase chain reaction-based study

Purpose: To determine the presence of herpesvirus DNA in the aqueous humor (AH) of patients with serpiginous choroiditis using polymerase chain reaction (PCR). Methods: AH from nine patients previously diagnosed with serpiginous choroiditis were investigated for herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV) by conventional virological methods and PCR. The PCR-positive DNA was gel-purified, extracted, and sequenced using a dye-based Applied Biosystems procedure. The sequences were processed through the National Cancer Institute’s BLAST inquiry for species identification. Results: Culture and cytological examination of AH from all nine patients were negative for HSV,VZV, and CMV. Five were positive for VZV, one was positive for HSV, and three were wholly negative using PCR. Subsequent DNA sequencing of the positive samples authenticated the presence of VZV and HSV DNA in the respective patients. Conclusion: VZV and HSV DNA were detected in a subset of patients with serpiginous choroiditis, suggesting that these viruses may function in the pathogenesis of this disease.     

Polymerase chain reaction in the diagnosis of herpetic keratitis: experience in a developing country

BACKGROUND: Herpetic ocular disease is a major cause of blindness. Rapid and accurate diagnosis is essential for prompt, proper treatment. We evaluated the usefulness of detection of herpes simplex virus (HSV) DNA by polymerase chain reaction (PCR) in the laboratory diagnosis of herpetic keratitis. METHODS: A retrospective study was conducted involving 234 patients who attended the cornea clinic at the Regional Ophthalmic Institute, Chennai, India, between March 1995 and September 1997. Inclusion in the study was based on clinical diagnosis of herpetic keratitis. Oligonucleotide primers directed against the HSV-I thymidine kinase gene were used, yielding a 110 base pair amplicon. The utility of PCR analysis was assessed against other diagnostic markers: HSV isolation on cell culture, HSV antigen detection by indirect immunofluorescence, detection of anti-HSV IgG by enzyme-linked immunosorbent assay (ELISA) and detection of HSV-specific tear secretory IgA (sIgA) by ELISA. These tests showed overall sensitivity values of 22.4%, 39.8%, 30.4% and 20.3% respectively. RESULTS: In epithelial keratitis all 35 specimens from which virus was cultured were positive by PCR. PCR gave a positive result in 23 (82.1%) of the 28 specimens in which HSV antigen was detected and in 4 (57.1%) of the 7 specimens that showed HSV-specific IgG. In addition, PCR detected HSV DNA in 5 of the 30 cases in which these three tests gave a negative result. PCR of two pooled tear samples (collected 1 week apart from the same patient) from 40 patients with stromal keratitis gave a positive result in 12 cases (30%). In stromal keratitis the sensitivity of PCR in detecting HSV DNA in tear samples was 85.7% with culture, indirect immunofluorescence and detection of anti-HSV IgG as the gold standard, and 80% with detection of sIgA as the gold standard. INTERPRETATION: The results confirm the good correlation with the clinical picture that can be obtained with PCR analysis. They also highlight the diagnostic utility of PCR in detecting HSV DNA in tear samples. This is particularly important in herpetic stromal keratitis, in which collection of corneal scrapings is not advised and, hence, conventional techniques such as virus isolation and antigen detection become difficult.     

Polymerase chain reaction and Goldmann-Witmer coefficient analysis are complimentary for the diagnosis of infectious uveitis

PURPOSE: To determine the relative contribution of the analysis of intraocular antibody production and the polymerase chain reaction (PCR) in aqueous humor (AH) to the diagnosis of infectious uveitis. DESIGN: Retrospective case-control study. METHODS: Paired AH and serum samples from 230 patients suspected of infectious uveitis were examined for intraocular antibody production against herpes simplex virus (HSV), varicella zoster virus (VZV), and Toxoplasma gondii by calculating the Goldmann-Witmer coefficient (GWC). In addition, AH samples were investigated by real-time PCR to determine the presence of microbial DNA. RESULTS: Positive results were obtained in 54 cases (23%): 13 HSV (24%), 16 VZV (30%), and 25 T gondii (46%). Of these, 23 (43%) were positive for both GWC and PCR, 26 (48%) only for GWC, and 5 (9%) only for PCR. With PCR as the sole diagnostic approach, a correct diagnosis of the infectious etiology would have been missed in 34% of cases for the herpes viruses and in 64% for T gondii. Analysis of the relationship between a positive laboratory diagnosis and the time of sampling after onset of ocular disease demonstrated that intraocular antibody production was found throughout the course of the diseases. Viral DNA was more readily detected early in infection. In contrast, T gondii nucleic acid was not detected until 3 weeks after onset of ocular disease. CONCLUSIONS: Analysis of intraocular antibody production contributed considerably to the etiological diagnosis of infectious uveitis, most notably of ocular toxoplasmosis early after onset of disease. Therefore, both PCR and GWC determination might be performed for comprehensive diagnosis of intraocular infections.     

Rapid detection of herpes simplex virus (HSV) antigen in human ocular infections

The new HERPCHEK (Dupont, No. Billerica, MA) enzyme immunosorbent assay (EIA) was used in a double-blind clinical study for rapid and specific detection of ocular herpes simplex virus (HSV) infection. This 4-hour assay can be used to demonstrate conclusively the presence of HSV antigen without culture and thereby rapidly differentiate between HSV and other clinically similar ocular infectious diseases. Ocular samples were collected from 180 individuals including 30 patients with acute HSV, 90 with latent HSV (ie, currently asymptomatic but with a positive history), 11 with acute or latent varicella zoster virus, 30 with nonherpetic infections (due to adenovirus, Acanthamoeba or bacteria), and 19 normal controls. A clinical diagnosis was made by one of us (DPL) and duplicate tear-film samples obtained by swabbing the conjunctival cul-de-sac and cornea. Coded samples were tested by routine viral culture on Vero cell monolayers and also were run independently in the HERPCHEK test. During active HSV infection, the HERPCHEK correlated 100% with clinical diagnosis, and virus culture correlated 90% with clinical diagnosis. In all latent HSV ocular infections, other nonherpetic ocular infections and normal samples, both the HERPCHEK and culture assays were negative.     

Detection of herpes viral genomes in normal and diseased corneal epithelium

Herpetic ocular disease is one of the major causes of corneal blindness. Clinical diagnosis of corneal disease is based principally on corneal appearance. However, abnormal morphology of the corneal epithelium (CE) is not an indicator for the presence of a herpes virus. Further, it has not been established if herpes viruses are present in normal corneal epithelial tissue. In these studies, the polymerase chain reaction was used to evaluate normal and diseased corneal epithelium for the presence of herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) genomic sequences. Thirty-two normal corneal epithelium specimens obtained from cadavers shortly after death were analyzed for HSV-1, EBV and CMV genomic sequences. Three of the 32 normal CE specimens were positive for amplified EBV DNA, 1 was positive for HSV-1 DNA, and none was positive for CMV DNA. We also tested eight herpetic dendritic lesions of which 3 were HSV-1 culture and PCR positive. The remaining five dendritic lesions were HSV-1 culture and PCR negative. Since these lesions were not evaluated for other herpesviruses, the etiology of these dendritic lesions is unknown. Six corneal epithelium samples from HIV-infected donors were negative for EBV, CMV and HSV-1 amplified sequences. Positive EBV, CMV and HSV-1 serology on all normal donors and on donors with clinically apparent disease did not correlate with positive PCR results. The results of these studies suggest that EBV and HSV-1 DNA can be amplified from a small percentage of apparently normal corneal epithelium.     

Infectious uveitis in immunocompromised patients and the diagnostic value of polymerase chain reaction and Goldmann-Witmer coefficient in aqueous analysis

PURPOSE: To establish the causes of uveitis in immunocompromised patients and to determine the contribution of polymerase chain reaction (PCR) and Goldmann-Witmer coefficient (GWC) analysis of aqueous humor in patients with an infectious etiology. DESIGN: Retrospective case series of 56 consecutive immunocompromised patients with uveitis. METHODS: All patients underwent full ophthalmologic examination and laboratory blood analysis for uveitis. Aqueous humor analyses were performed using PCR and GWC for cytomegalovirus (CMV), herpes simplex virus (HSV), varicella zoster virus (VZV), and Toxoplasma gondii. RESULTS: Of 56 immunocompromised patients, 43 (77%), all posterior and panuveitis, had intraocular infections. Twenty-one (49%) had CMV, three (7%) had VZV, 11 (26%) had T. gondii, six (14%) had Treponema pallidum, and one (2%) each had Aspergillus and Candida. In AIDS patients, CMV was the most common cause. A strong correlation between AIDS and ocular syphilis was also observed (P = .007). In nonAIDS immunocompromised patients, T. gondii was most frequently detected. Twenty-seven patients were examined by both PCR and GWC; five (18.5%) were positive by both assays, 15 (55.5%) were positive by PCR alone and seven (26%) by GWC alone. Viral infections were detected by PCR in 16 of 17 (94%) cases; T. gondii in four of 10 (40%) patients. Using GWC, a viral infection was diagnosed in three of 17 (18%) and T. gondii in nine of 10 (90%) cases. CONCLUSIONS: In immunocompromised patients, PCR is superior in diagnosing viral infections. Analysis of intraocular antibody production played a decisive role in the diagnosis of ocular toxoplasmosis.