British Journal of Ophthalmology 1996; 80: 235-240

ORIGINAL ARTICLES - Laboratory science

Diagnostic assays in cytomegalovirus retinitis:detection of herpesvirus by simultaneousapplication of the polymerase chain reaction andlocal antibody analysis on ocular fluid

Peter Doomenbal, G Seerp Baarsma, Wim G V Quint, Aize Kijlstra,Philip H Rothbarth, Hubert GM Niesters

AbstractAim-To determine the value of the poly-merase chain reaction (PCR) techniqueand the analysis ofintraocularly producedantibodies by calculating a Goldmann-Witmer quotient (GWq) as diagnosticassays in the confirmation of a clinicallydiagnosed cytomegalovirus (CMV) retini-tis in a group ofunselected AIDS patients.Methods-Eleven samples of undilutedocular fluid, obtained from nine AIDSpatients with a clinically diagnosed CMVretinitis were analysed for the presence ofgenomic DNA from CMV, HSV-1, VZV,and EBV by PCR. Nine of these sampleswere analysed for the presence of locallyproduced IgG antibodies against theseherpesviruses by calculating a GWq. Tensamples obtained from patients withvarious entities of clinical non-herpeticuveitis and 17 samples ofaqueous humourobtained at cataract surgery were used ascontrols.Results-In 10 out of 11 samples fromAIDS patients (91%) the presence ofCMVDNA was demonstrated. In four out ofnine (44%/o) patients this was accompaniedby CMV DNA in the blood indicating aCMV viraemia. In one sample, VZV DNAwas detected and in another sample bothCMV and VZV DNA were detected. NoHSV-1 or EBV DNA could be demon-strated in these 11 samples. In contrast,simultaneous analysis of locally producedIgG antibodies against herpesvirusescould not confirm the initial diagnosis ofCMV retinitis. Ocular fluid samplesobtained from 10 control uveitis patientswere negative for DNA from CMV, VZV,and EBV by PCR. In one of 10 uveitis con-trol samples HSV-1 DNA was detected;antibody analysis did not confirm this. Inthe uveitis control group, a significantGWq was calculated in one sample forHSV-1 and in another sample for VZV.The cataract control samples were allherpesvirus DNA negative by PCR.Conclusions-To establish the diagnosisof CMV retinitis in AIDS patients,

ophthalmoscopic examination is a sensi-tive method. In confirming a diagnosis inindistinctive cases, application of a PCRassay detecting CMV DNA is a moresensitive method than analysis of locallyproduced antibodies by calculating a GWq.In clinical non-herpetic uveitis, secondaryrelease ofHSV-1 and VZV should be con-sidered requiring additional therapeuticanticipation.(BrJ7 Ophthalmol 1996; 80: 235-240)

Cytomegalovirus (CMV) has been recognisedas a causative agent in the diagnosis of uveitisby virus culture for 35 years.' CMV retinitismainly affects immunocompromised patientsand predominantly AIDS patients.2-8 Onlythree cases of CMV retinitis have beenreported in apparently healthy individuals.6 9 10

Clinically, the patients' main complaintsare blurred vision, scotoma, and progressiveimpaired visual acuity, but asymptomaticocular infections also occur. The extent of thesymptoms depends on the location and typeof tissue damaged by the inflammatory reac-tion.5 6 11 Early ophthalmoscopic findingsinclude diffuse granular infiltrates that maymimic cotton wool spots. Later, typicalgranular haemorrhagic lesions with centri-fugal spread, yellow white perivascular infil-trates, retinal oedema, and vascular sheathingcan occur.2 11 Finally, atrophy of the affectedretina and the pigment epithelium andfibrosis may become apparent.

In most cases the diagnosis of CMV retinitisis based on clinical examination and findingsby ophthalmoscopy which are typical but notalways specific. The differential diagnosis ofCMV retinitis should include non-infectiouscotton wool spots, toxoplasmosis, candidiasis,syphilis, herpes simplex virus (HSV-1), andvaricella zoster virus (VZV) retinitis.8 12 InAIDS patients, the diagnosis can be masked bymultiple agents co-infecting the retina. Foran adequate choice of therapy confirmation ofthe clinical diagnosis of CMV retinitis bylaboratory tests might be required.3 13

Conventional culture of CMV from blood,

The Eye Hospital,RotterdamP DoornenbalG S Baarsma

Department ofVirology, UniversityHospital Dijkzigt,RotterdamP H RothbarthH GM Niesters

Department ofMolecular Biology,Diagnostic CenterSSDZ, DelftW G V Quint

The NetherlandsOphthalmic ResearchInstitute, AmsterdamA Kijlstra

Correspondence to:P Doornenbal, MD, The EyeHospital, PO Box 70030,3000 LM Rotterdam, theNetherlands.Accepted for publication13 September 1995

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aqueous, or vitreous fluid can take a long timeto grow and it is rarely positive, limiting itsconsequences for treatment.2 5 A positiveblood culture, however, can be helpful inestablishing a disseminated CMV infection,which is accompanied by CMV retinitis in22-48% ofAIDS patients.4 1114The most sensitive method in antibody

production analysis in uveitis patients, iscalculation of a Goldmann-Witmer quotient(GWq). 15 16 This method determines the ratioof antibody titres against suspected intraocularpathogens in ocular fluid and serum to thetotal amount of IgG and is helpful in confirm-ing the diagnosis of toxoplasma uveitis andacute retinal necrosis syndrome.15-20 In AIDSpatients, the detection ofCMV DNA using thepolymerase chain reaction (PCR) techniquemight be more suited to confirm a clinicalCMV retinitis than local antibody productionanalysis since the PCR technique does notdepend upon an immunological response ofthe host, which is impaired in the later stagesof AIDS.21 Several authors have reportedherpesvirus DNA detection in ocular fluids ofpatients with an active CMV retinitis using thePCR technique.22-24

Assays based on the PCR are yet to be intro-duced in routine diagnostics of uveitis butbefore its general use can be accepted, thevalidity of the technique and the interpretationof the test results have to be analysed. Thepurpose of this study was to determine thevalue of the PCR technique as a diagnostic toolin confirming the diagnosis of CMV retinitiscompared with the analysis of locally producedantibodies by calculating a GWq for ocularfluids of AIDS patients with a clinicallydiagnosed CMV retinitis under antiviraltherapy.

Materials and methods

PATIENTSWith the patients' informed consent andaccording to the World Medical Associationdeclaration of Helsinki, 1964, undiluted ocularfluid samples of two groups of patients werecollected during therapeutic pars plana vitrec-tomy or by diagnostic anterior chamber para-centesis in case of a clinically undeterminedfeature of uveitis. The diagnosis of CMVretinitis and uveitis was based on the pre-viously described features and made by twoophthalmologists experienced in uveitis. Thediagnosis of AIDS was made according to thedefinition ofthe Centers for Disease Control.25All patients had serum anti-HIV antibodies,had suffered from multiple opportunisticinfections, and had CD4+ cell counts less than400X 106/1.

In group A, 11 samples of undiluted ocularfluid from 11 eyes were obtained from nineconsecutive, unselected AIDS patients with aclinical CMV retinitis. At an inactive stage,eight samples of vitreous fluid and one sampleof subretinal fluid were obtained at vitrectomyrequired for retinal detachment. Aqueoushumour was obtained from two eyes for

diagnostic purposes at an active stage of thedisease. The samples were directly split intotwo portions. One was used for antibodyanalysis and the other was used for DNAisolation. Serum samples for antibodyanalysis were obtained from all nine AIDSpatients.

In group B, 10 split samples of undilutedocular fluid obtained from 10 patients with theclinical features of an active uveitis not suspectfor herpetic aetiology served as controls.Group B included patients with a low gradeendophthalmitis (n=3), Behcet's disease,uveitis posterior (n=3), toxoplasma uveitis,non-Hodgkin's lymphoma associated uveitis,and a candida retinitis. Vitreous fluid sampleswere obtained from eight patients and aqueoushumour samples were obtained from twopatients. Serum samples were obtained fromall 10 patients. Additionally, 17 samples ofaqueous fluid obtained at cataract surgery ofotherwise healthy individuals were onlyanalysed by PCR assays. To establish apossible CMV viraemia an EDTA bloodsample was collected from all patients andanalysed for the presence of CMV DNA byPCR.

DNA EXTRACTION AND PCRTo enhance the efficacy of the PCR assay wepurified the target DNA from both ocular fluidand blood samples. 14 26 27 Briefly, DNA wasextracted from 100 ,ul of undiluted ocular fluidstored in 1 ml lysis buffer A (4 M guanidine-iso-thiocyanate/0- 1 M Tris-HCl pH 6 4/44mM EDTA/2-5 ml per 100 ml Triton X-100).28 A total of 40 RI Celite solution (2 g/ml,Jansen Chemika, the Netherlands) was addedand the suspension was incubated for 10minutes at room temperature to adsorb theDNA. The Celite pellet was washed twice withlysis buffer B (4 M guanidine-isothio-cyanate/0 1 M Tris-HCl pH 6 4), twice with70% ethanol, and once with acetone. The pel-let was dried for 15 minutes in a vacuum exsic-cator and resuspended in 200 [LI water at56'C. After 2 minutes of centrifugation(15 000 g), 180 ,ul of the supematant contain-ing the DNA was collected.A volume of 500 ,ul of the EDTA blood

samples was suspended in 9*5 ml lysis bufferA. The DNA was isolated, using 300 ,ul ofthis suspension, by means of a phenol/chloroform/isoamyl alcohol extraction andethanol precipitation. The DNA pellet wasresuspended in 200 ,u water.The PCR assays were performed detecting

DNA from CMV, HSV-1, VZV, Epstein-Barrvirus (EBV), and a human 1 globin sequence.The PCR assay detecting the human 1 globinsequence was used to verify the integrity of thetarget DNA and the yield of extraction method.The sequences of the primers and probes forCMV, HSV-1, VZV, and ,B globin have beenpublished previously.24 2932 For detection oftheHSV-1 DNA, the outer primers were used. Forthe CMV primers we used a single base modifi-cation ofthe originally published sequence ofthe3'-primer in which at the 3'-terminal end, a

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Table 1 Clinical data ofcytomegalovirus (CMV) retinitis patients (group A) and control uveitis patients (group B)

Patient Sex, Ocular First ocular Medication CD4+ cellsNo age (years) Initial diagnosis fluid appearance* started* Medication (X 106/7)

Group A:1 M, 61 CMV retinitis Aqu 2 3/4 Ganciclovir/foscarnet 302R M, 40 CMV retinitis Vit 5 5 Ganciclovir 102L CMV retinitis Vit 1 6 Ganciclovir 103 M, 35 CMV retinitis SRF 5 5 Ganciclovir 404 M, 38 CMV retinitis Vit 7 7 Ganciclovir <4005 M, 42 CMV retinitis Vit 2 2 Foscarnet/acyclovir 806 M, 36 CMV retinitis Vit 5 5 Ganciclovir/acyclovir 107 M, 29 CMV retinitis Aqu '/2 1/4 Ganciclovir 208R M, 50 CMV retinitis Vit 6 6 Foscarnet 108L CMV retinitis Vit 6 6 Foscarnet 109 F, 34 CMV retinitis Vit 2 2 Ganciclovir <10

Group B:1 M, 75 Low grade endophthalmitis Vit 18 Periodic Steroids/antibiotics -

2 F, 72 Candida retinitis Vit 1/4 Continuous/'/4 Steroids/antimycotics -

3 F, 57 Low grade endophthalmitis Vit '/2 '/4 Antibiotics -

4 M, 66 Uveitis posterior Vit 5 Periodic Steroids -

5 M, 26 Uveitis posterior Vit 24 Periodic Steroids -

6 F, 43 Low grade endophthalmitis Vit 8 Periodic Steroids -

7 F, 58 Non-Hodgkin's retinitis Aqu 2 - Radiotherapy -

8 F, 29 Uveitis posterior Vit 18 Periodic Steroids -

9 M, 53 Toxoplasma uveitis Aqu 2 2/1/4 Steroids/pyrimethamine -

10 M, 37 Behqet's disease Vit 12 - Photolaser coagulation -

*Months before sampling. Aqu=aqueous fluid; Vit=vitreous fluid; SRF=subretinal fluid; -=not done.

guanidine base was replaced by a cytosine base(3'-primer CGTTTGGGTTGCGCAG-C-GGG).24 The sequences of the primers andprobe detecting the BamHI-W fragment of theEBV genome are: 5'-primer CTCTCTCT-GTCCTTCAGAGG, 3'-primer GTGGCTC-CCCTCAGACATTC, and the probeAACCAGGGACCTCGGGCACCCCA-GAGCCCC.A total volume of 100 ,ul reaction mix con-

tained 10 ,ul of the DNA isolate, 50 pmol ofeach primer, 200 puM ofeach desoxynucleotidetriphosphate, 50 mM KC1, 10 mM Tris-HClpH 9 0, 2-5 mM MgCl2, 0-01% gelatine, 01%Triton X-100, and 1 U Taq DNA- polymerase(Promega). The PCR was processed using aThermocycler 60/2 (Biomed, Germany). Theprocessing conditions for all assays were pre-heating for 4 minutes at 94°C, denaturation for1 minute at 94°C, annealing for 1 minute at52°C, and elongation for 1 minute at 74°C,repeating a total of 40 cycles except for theCMV assay which was annealed for 30 secondsat 48°C. Twenty five pul of the amplimers wereelectrophoresed on a 3% agarose gel. The gelwas electroblotted to a HybondN+ membrane(Amersham) at 3-5 mA/cm2 for 35 minutes.The membranes were hybridised with 32pATP-labelled oligonucleotides. After overnightincubation at 37°C, the membrane was washedtwice for 15 minutes with 2XSSC/0-1% SDSand once with 0-5XSSC/0d1% SDS for 15minutes at 56°C (1XSSC is 15 mM sodiumcitrate, 150 mM NaCl). Finally, an autoradi-ographic film was exposed for 4-5 hours.A sample of double distilled water going

through the extraction procedure and thePCR, and a second sample of double distilledwater only going through the PCR served asnegative controls. DNA extracted from celllines infected with CMV, HSV-1, VZV, orEBV served as positive controls.

ANTIBODY ANALYSISAnalysis ofIgG class antibody titres against theherpesviruses on a paired serum and ocularfluid sample was performed by using a fixed

cell immunofluorescence technique as des-cribed by Luyendijk et al.20 The total amountof IgG in serum and ocular fluid was deter-mined by a radial immunodiffusion technique.Antibodies were detected on twofold serialdilutions of the ocular fluid samples and of theserum samples. A GWq was calculated andconsidered significant if above 3 0.

ResultsGroup A included nine AIDS patients with theclinical diagnosis of CMV retinitis (Table 1).Their mean age was 41 ( range 29-41) years.The mean duration of ocular symptoms untilobtaining the ocular fluid sample was 3-8(range l/2-7) months. Eight patients were diag-nosed having an unilateral CMV retinitis andone patient was diagnosed having a bilateralCMV retinitis. In one patient (A8, Table 1) abilateral CMV retinitis became apparent 1month after obtaining the sample from the firstaffected eye. All nine patients in group A hadreceived antiviral therapy before obtaining thesample with a mean of 4-0 months (range 4days-6 months). All AIDS patients had CD4+counts less than 50 cells X 106/1 except forpatient A4, who was suffering from AIDS andnon-Hodgkin's lymphoma, and patient A5.The patients of the control group B had a

mean age of 52 (range 26-75) years (Table 1).They did not receive antiviral therapy during thecourse of disease. Patient B2 had receivedcontinuous steroid therapy. Five patients (B 1,B4, B5, B6, B8, Table 1) intermittently receivedsteroid therapy in case of exacerbation of theocular inflammation. One patient, B7, wastreated with radiotherapy and patient B8received pyrimethamine, an antiprotozoic agent.

Eleven ocular fluid samples of group Apatients were analysed for the presence ofDNA sequences of the herpesviruses and thehuman P globin gene (Table 2). In 10 of 11samples CMV DNA was detected. One samplewas positive for both CMV and VZV DNA(A5) and another (A6) was only positive forVZV DNA. Genomic DNA from EBV orHSV-1 was not detected in any of the 11

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Table 2 Summary of the results of the polymerase chain reaction assays and Goldmann-Witmer quotients on ocularfluids and serum samples ofgroup A and group B

CMV Ocularfluid Goldmann-Witmer quotient*Patient DNA DNA positiveNo in blood for CMV HSV-1 VZV EBV

Group A:1 - CMV <3 - - -2R Pos CMV 1.0 1.0 1 0 1.02L Pos CMV - - - -3 - CMV - - - -

4 Pos CMV <1 <1 <1 <15 - CMVNZV 1.0 1-2 <1 -6 - VZV 1-2 <1 <1 1-27 Pos CMV 40 8-0 31-9 <18R Pos CMV <1 <1 <1 <18L Pos CMV <1 <1 <1 <19 - CMV -t <1 <1 1.1

Group B:1 - HSV-1 <1 1-2 <1 -2 - - <1 6-4 -t 103 - - - 1.1 8-4 -4 - - 1-8 0 <1 <15 - - - 1-6 -t -t6 - - - <1 -t -t7 - - 19 <1 <1 <18 - - -t 1-6 -t -t9 - - -t <1 <1 <110 - - -t 2-7 2-7 2-7

CMV=cytomegalovirus; HSV=herpes simplex virus; VZV=varicella zoster virus;EBV=Epstein-Barr virus.*A Goldmann-Witmer quotient of 3 0 or more is considered significant. tNot calculable owingto a negative antibody titre in serum and/or ocular fluid. -=Not done. R=right eye; L=left eye.

samples. In all samples the human P globinDNA sequence could be detected.The blood samples of the CMV retinitis

patients showed detectable CMV DNA in fourpatients (A2, A4, A7, A8, Table 2). In twopatients, two blood samples were examined forCMV DNA over a time span of 1 month.In both cases the first and second samplewere CMV DNA positive despite antiviralmedication.The ocular fluid samples of the control

group B showed no detectable CMV, VZV orEBV DNA (Table 2). In one of 10 samples,HSV-1 DNA was detected (Bi). No CMVDNA was detected in the whole blood samplesof the control group B. In the aqueous of the17 cataract patients no herpesvirus DNA wasdetected. In all control samples the 3 globinDNA was detected.

Table 3 Serological data on ocularfluid samplesfrom cytomegalovirus (CMV) retinitispatients, group A and uveitis control patients, group B

Serum antibody titre (I:titre) Ocularfluid antibody titre (I:titre)PatientNo CMV HSV-1 VZV EBV CMV HSV-1 VZV EBV

Group A:1 64 1024 256 2048 <4 - - -2R 128 1024 512 256 8 64 16 162L - - - - - - - -

3 - - - _ _ _ _ _4 256 1024 1024 2048 16 128 128 5125 1024 1024 128 - 32 64 4 -6 64 2048 64 1024 16 256 8 2567 256 512 256 1024 2 8 16 28R 256 2048 512 512 8 64 16 168L 256 1024 1024 4096 16 64 16 649 16 2048 256 64 0 32 4 2

Group B:1 1024 1024 128. - 32 64 4 -2 512 16 - 256 8 2 0 163 - 64 16 - - 2 4 -4 64 <16 256 1024 8 0 16 645 - 2048 256 256 - 8 0 06 - 1024 256 256 - 128 0 07 64 1024 64 64 8 4 4 648 32 2048 1024 1024 0 8 0 09 16 256 1024 64 0 8 8 210 256 64 256 256 0 2 8 8

HSV=herpes simplex virus; VZV=varicella zoster virus; EBV=Epstein-Barr virus.-=Not done. R=right eye; L=left eye.

All the analysed sera and the ocular fluidsamples of group A patients had positive anti-body titres against HSV-1, VZV, and EBV(Table 3). In eight of nine (89%) tested ocularfluid samples, IgG anti-CMV antibodies weredetected. The GWq was calculated on ninesamples of ocular fluid of group A for CMV,HSV-1, and VZV. A GWq for EBV was calcu-lated on seven samples. In sample A7, a signifi-cant GWq was found for CMV as well asfor HSV-1 and VZV (Table 2). None of thesamples of group A had a significant GWq forany of the tested herpesviruses althoughintraocular antibodies against herpesviruseswere present (Table 3).

In the control group B, paired serum andocular fluid samples were obtained from all 10patients. Patient B4 had a serum titre lowerthan 1:16 for HSV- 1 (Table 3). All otherpatients tested had positive serum antibodytitres for all herpesviruses - that is, equal orlarger than 1:16. In group B, four of seven(57%) ocular fluid samples tested for CMVhad a positive antibody titre. For HSV-1 thiswas nine of 10 (90%/o), for VZV six of 10(60%), and for EBV this was five of eight(63%). No sample of the control group Bshowed a significant GWq for CMV andEBV. One sample (B2) showed a significantGWq for HSV- 1. Another sample (B3)showed a significant GWq for VZV(Table 2).

DiscussionThis report compares the results of analysis oflocal antibody production against herpes-viruses and PCR assays, detecting DNA ofherpesviruses in ocular fluid, in relation tothe clinical diagnosis of CMV retinitis inunselected cases in AIDS patients. The clinicaldiagnosis could not be confirmed in nine of thetested cases by a significant GWq. Comparedwith the PCR assays the clinical diagnosis ofCMV retinitis showed a sensitivity of 100%and a specificity of 91%.

In comparing both types of assays oneshould consider the basic differences in tech-nique. The PCR assay is a test, detectingdirectly a part of the virus. The analysis of anti-body production, however, is an indirect test,depending on an immunological response ofthe host against the virus, which is normallyreflected in a rise of the antibody titre, butaffected in AIDS patients.The results of the very specific and sensitive

PCR technique require a well balanced inter-pretation, especially in analysing viruses of theherpes family such as CMV, HSV-1, VZV, andEBV. These viruses can establish latent infec-tions in humans and since all patients testedhave detectable serum IgG antibodies againstall herpesviruses, except patient B4, who hadan antibody titre less than 1:16 for HSV-1,they are expected to be carriers of theseviruses. However, the aqueous samples of 17cataract patients were all negative for herpes-virus DNA, indicating that normally noherpesvirus is present in ocular fluid ofnon-inflamed eyes.

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An inflammatory reaction as in uveitis canbe a trigger for secondary release of latentherpesviruses and lead to clinically falsepositive PCR results if detection of theprimary pathogen is intended. In ocular fluidsfrom the uveitis control patients, no DNAfrom the neurotrophic latent VZV or thelymphocytotrophic latent EBV and CMV wasdetected. This indicates that no positivePCR results were caused by herpesvirusesreleased from latently infected sensorynerve endings, lymphocytes, or vascular endo-thelium cells.

In group A the role ofVZV as a co-infectantor primary pathogen was anticipated. Inpatient A5, for instance, both CMV DNA andVZV DNA were detected. The ophthalmo-scopic features were, however, indicative of aCMV retinitis and lacked a pronounced retinalnecrosis. Sample A6, on the other hand, wasCMV DNA negative but VZV DNA was posi-tive. In this case, ophthalmoscopic and clinicalfeatures were indecisive. The results of thePCR assays indicated an acute retinal necrosissyndrome was more likely and after additiveacyclovir therapy the process was haltedtemporarily. In one uveitis control sample(B1), HSV-1 DNA was found and secondaryHSV release must be considered. This patientwas suffering from a recurrently eliciting irido-cyclitis and a low grade endophthalmitis for1-5 years after a cataract extraction with lensimplantation. Initially, the inflammationresponded well to antibiotic and steroidtherapy. Later, removal of the implant lens andvitrectomy were necessary. Bacterial culturesof the vitreous fluid were negative and theGWq for CMV, HSV-1, and VZV wereinsignificant. The insignificant GWq forHSV-1 can be due to wrong time of samplingor to extinction of the immune response afterfrequent stimulation leading to an absence inrise in antibody production, especially of theIgG class, as has been documented in patientsrepeatedly suffering from recurrent herpeticinfections.'8 33

In AIDS patients a CMV viraemia withleakage of viral particles through the blood-retinal barrier might lead to clinically falsepositive PCR results in ocular fluid. Toexclude this, detection of CMV DNA inblood served as a control. Five out of nineblood samples of group A were CMV DNAnegative indicating that CMV DNA in thepaired ocular fluid did not originate from theblood. Regarding the paired samples beingobtained several weeks after onset of theCMV retinitis the results do not excludeintraocular CMV particles originating from aprevious viraemia. It is, however, remarkablethat despite prolonged intravenous antiviraltherapy four blood samples were CMV DNAnegative whereas the corresponding ocularfluid samples were positive and retrospec-tively confirming the clinical diagnosis. Sinceclearance of the virus is not only dependingon virustatic therapy but also on a functionalimmune response, the absence ofCMV DNAin these blood samples might reflect a residualimmune function (three of the four patients

had CD4+ counts between 30 and 80 cellsx 106/1) combined with a poor penetration ofthe antiviral medication through the blood-retinal barrier. Resolution of detectable CMVDNA in blood after antiviral therapy has beenreported earlier.'4

Control uveitis samples showed no signifi-cant GWq for CMV and thus no false positiveresults were found. In group A also no confir-mation of the initial diagnosis ofCMV retinitiswas demonstrated by a positive GWq.Although minimal use of antibody based assaysin AIDS patients might be expected, all butone ocular fluid sample of AIDS patientsproved to contain CMV DNA and anti-CMVantibodies, and all sera were anti-CMV IgG orCMV DNA positive or both. Theoretically aninconclusive GWq must result from the insuf-ficient absolute or relative rise of local antibodyproduction. In group A cases an insufficientabsolute rise of local antibody productionagainst CMV is likely to be caused by animpaired antibody production as has beendescribed in the later stage of AIDS, bothin quantity and quality, and in cytotoxicreaction.2' This can be expected because of thelow CD4+ counts. An insufficient relative risein local antibody production can result fromleakage of antibodies through the blood-retinalbarrier due to a destructive inflammatoryreaction. Furthermore, an overwhelmingpresence of antigen in the eye might capturethe intraocular antibodies, restraining themfrom analysis. The ratio of antibody titres incalculating a GWq can further be disturbed byrelatively high serum IgG antibody titresagainst CMV, caused by an actual or recentCMV viraemia and high total IgG levels, as isfrequently observed in AIDS patients.2'Confusingly, sample A7 showed a significantGWq for CMV, HSV-1, and VZV whereasonly CMV DNA was found. Here a possiblyaspecific polyclonal antibody production or aco-infection might have led to an indecisiveGWq.18Two samples of the control group (B2, B3)

showed a significant GWq, indicating apossible pathogenic role for these viruses.Patient B2 showed a significant GWq forHSV-1 but the PCR assay was negative. Thispatient was suffering from a systemic lupuserythematosus and was treated continuouslywith prednisone. A vitrectomy was performedand vitreous fluid was cultured and foundpositive for Candida tropicalis, whichresponded well to antimycotic therapy. Asignificant GWq for VZV was found in apatient (B3) suffering from a low gradeendophthalmitis for 1 month after cataractextraction with lens implantation whichresponded well to antibiotic therapy. Vitreousfluid was culture positive for Staphylococcusepidermidis. A relapse 3 weeks after surgery wastreated successfully with acyclovir as aqueousshowed a significant GWq for VZV. A secondrelapse occurred within a week after cessationof acyclovir therapy. Removal of the intra-ocular implant lens prevented further relapses.The absence of viral DNA seems similar to thenegative PCR results in the course of herpes

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simplex encephalitis, perhaps as a result of latetiming of obtaining the sample, especiallywhen it concerns immune competent patientsbasically capable of clearing the virus.34

In conclusion, ophthalmoscopic examinationis a sensitive method for the clinical diagnosis ofCMV retinitis in unselected cases in AIDSpatients. In indistinctive cases confirmation ofthe diagnosis of CMV retinitis by detectingCMV DNA in ocular fluid using a PCR assay isa more sensitive tool than analysis of locallyproduced antibodies. The herpesvirus PCRassays can support the choice of therapy inatypical clinical cases. In the case of a clinicalnon-herpetic, active uveitis the' role ofsecondary released HSV-1 or VZV should beconsidered, possibly requiring additional thera-peutic anticipation. Since no postoperativecomplications were observed, anterior chamberparacentesis is a safe method to obtain clinicalsamples for laboratory analysis.

In future studies, the value of PCR assays forother aetiological agents in infectious uveitisand other patient groups must be established.Moreover, efforts should be made to enhancethe efficacy of the PCR assays in ophthal-mology, and to develop standardised protocolsand intercentre quality control programmesallowing better comparison of the results ofdifferent studies.

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