Genitourin Med 1995;71:35-40

Clinico-Pathological Conference

Progressive multifocal leukoencephalopathy inpatients with AIDS: detection ofJC virus DNA inCSF and brain

C J Perrons, R J S Chinn, J D Fox, S B Lucas, M J G Harrison, R F Miller

Division of Virology,Department ofMedical MicrobiologyC J PerronsJ D FoxDepartment ofHistopathologyS B LucasDepartment ofNeurologyM J G HarrisonDepartment ofMedicine, UniversityCollege LondonMedical School,London WClE 6JJR F MillerMRI Unit,Department ofImaging, MiddlesexHospital, LondonWlN 8AAR J S ChinnCorrespondence to:Dr R F Miller, Departmentof Medicine, UCLMS,Middlesex Hospital, LondonWIN 8AA, UK.Accepted for publication7 June 1994

Case presentations (Dr R F Miller)Case 1A 56 year old right handed Caucasian homo-sexual unemployed laboratory technician wasadmitted as an emergency in March 1993 viathe accident and emergency department, withthe sudden onset of slurred speech, left facialweakness and falling to the right. On specificenquiry he denied loss of consciousness,visual disturbances or headache; he feltgenerally weak and tired and had no othersymptoms.He was first found to be HIV-1 antibody

positive in April 1987 and participated in theMRC Concorde Study until March 1992, atwhich point he developed oral candidiasis anda mild peripheral sensory neuropathy: hisCD4 count had fallen to 190/mm.3 At thistime he began co-trimoxazole as primaryprophylaxis against pneumocystis pneumoniaand "open label" zidovudine. This lattermedication produced neutropenia and it wasstopped; he began dideoxyinosine (ddl).On examination he was orientated in time

and place but had a short attention span; hehad dysarthria but no dysphasia.Neurological examination revealed left sidedneglect, a left upper motor neuron seventhcranial nerve palsy and left sided weakness inboth arm and leg. There was an extensor leftplantar and also the palmar-mental and graspreflexes were both brisk (suggesting a frontallesion). Investigations revealed: haemoglobin= 12-7 g/dl, WBC = 4-1 (neutrophils = 3 3)x 109/1; Urea and electrolytes and liver func-tion tests were normal. Serum toxoplasmaand syphilis serology and cryptococcal anti-gen (CRAG) were negative. Culture of bloodwas negative for bacteria, mycobacteria andfungi.CT of the head showed a right frontal low

attenuation lesion without enhancement andwithout mass effect. At lumbar puncture thecerebrospinal fluid (CSF) was clear andcolourless; analysis showed protein = 1-82 g/l;no cells were seen. Gram, Auramine, Indiaink stains and culture for bacteria, myco-bacteria, fungi and viruses were negative.DNA amplification, using the polymerasechain reaction (PCR) for Herpes simplex virus1 and 2, cytomegalovirus (CMV) and vari-cella zoster virus DNA was negative.Empirical antitoxoplasma therapy was begunwith sulphadiazine and pyrimethamine, butthis produced a widespread maculopapularrash after 10 days of therapy and so it wasstopped. MRI was performed.

MRI scan (Dr R Jf S Chinn)This showed agenesis of the corpus callosum(an incidental finding). A focal region of highsignal intensity was seen in the right frontalwhite matter on T2 weighted imaging (fig 1).This extended out into the gyral cores sparingthe cortical grey matter. It also extended infe-riorally along the white matter tracts throughthe internal capsule and crus cerebri into theright side of the pons. This lesion was of lowsignal intensity on TI weighted imaging(fig 2). It did not enhance after the adminis-tration of intravenous gadolinium-DPTA.There was no mass effect associated with thislesion. There were further similar smalllesions in the right cerebellar hemisphere andin the right temporal lobe. These appearanceswere most likely to be due to progressivemultifocal leukoencephalopathy.

Dr R F MillerOn the basis of clinical presentation and MRIappearances it was planned to perform a brainbiopsy in order to confirm the diagnosis. Thepatient was initially in agreement with thisdecision but, following informed consent,declined surgery. His condition subsequentlydeteriorated rapidly with difficulty swallowingand recurrent aspiration of fluids. Terminally

Figure 1 Case 1: T2 weightedMRI scan showing highintensity signal in the right frontal white matter.

35

on June 4, 2020 by guest. Protected by copyright.

http://sti.bmj.com

/G

enitourin Med: first published as 10.1136/sti.71.1.35 on 1 F

ebruary 1995. Dow

nloaded from

Perrons, Chinn, Fox, Lucas, Hamison, Miller

Figure 2 Case 1: Tlweighted MRI scanshowing low intensitysignal (arrows) extendingalong the white mattertracts through the internalcapsule.

he was confused, disorientated and had a poormemory. A necropsy was performed.

Pathology (S B Lucas)At necropsy, the patient was not wasted.There were blisters on the trunk and a wide-spread maculopapular rash. Internally, bothtrachea and lower oesophagus were severelyinflamed, and the lungs (combined weight2230 g) were consolidated. The brain (weight1370 g) had the rare phenomenon of a con-

genitally absent corpus callosum (fig 3).Softening of the white matter was evident inthe right frontal lobe, anterior right thalamusextending to the right cerebral peduncle.

Histologically, the skin lesion was erythemamultiforme (due to a drug reaction). Whilstthe tracheitis was associated withcytomegalovirus (CMV) the oesophagitis hadno identifiable pathogen. There was alsoCMV adrenalitis. The lungs showed Grampositive coccal pneumonia. In the brain wide-spread progressive multifocal leukoen-cephalopathy (PML) was present not only inthe grossly abnormal areas but also in thecerebellar white matter (figs 4, 5). The spinalcord was normal.

Conclusions of necropsyMajor pathologies: 1. bacterial pneumonia, 2.progressive multifocal leukoencephalopathy,3. congenital absence of corpus callosum.

Figure 3 Case 1:Coronal slice of brain. Thecorpus callosum is absent.The abnormal whitematter ofprogressivemultifocalleukoencephalopathy is (asusual) difficult to depict inphotographs when it hasnot progressed tofranknecrosis.

Figure 4 Case 1: Progressive multifocalleukoencephalopathy in brain. The tissue is "empty" apartfrom microglial cells phagocytosing myelin debris. (x 400)(haematoxylin and eosin).

Minor pathologies: 1. CMV adrenalitis andtracheitis, 2. erythema multiforme due tosulphadiazine.

Case 2 (R F Miller)A 32 year old right-handed Caucasian homo-sexual financial consultant was admitted tohis local hospital in September 1993 with afour week history of clumsiness and weaknessof the right arm and weakness of the right sideof the face. In addition he reported poormemory and concentration; these symptomswere confirmed by both friends and family. Inthe two weeks before admission he hadnoticed progressive dysphasia and in additiondifficulty in writing. He denied headache, lossof consciousness, auditory or visual problemsand had no past history of note. He had livedand worked in the United States for the pastten years but more recently had lived andworked in Central Africa. He had multiplehomosexual partners and had engaged in highrisk sexual activity. He had never had an HIVtest.On examination at his local hospital, the

neurological signs were of a right upper motorneuron seventh cranial nerve and a righttwelfth nerve cranial palsy. There was nograsp or palmomental reflex. Tone was spasticand power was MRC grade 2 in the right arm.There was generalised hyper-reflexia withclonus of the right ankle. The plantar

Figure S Case 1: Progressive multifocalleukoencephalopathy in brain. Less severe lesion showingnumerous large bizarre astrocytes characteristic ofPML.(x 100) (haematoxylin and eosin).

36

. .I.

I "-Z .":. : . 1,

e.

..

...1.& I

I

-4

'... .. I

II

... 1. t

I I

. . :I

on June 4, 2020 by guest. Protected by copyright.

http://sti.bmj.com

/G

enitourin Med: first published as 10.1136/sti.71.1.35 on 1 F

ebruary 1995. Dow

nloaded from

Progressive multifocal leukoencephalopathy in patients with AIDS: detection ofJC virus DNA in CSF and brain

responses were bilaterally extensor.Investigations showed haemoglobin = 12-8g/dl, WBC = 4.7 (neutrophils = 4.3) x 109/1;urea and electrolytes and a chest radiographwere normal. CT of the head showed diffusecerebral white matter pathology. MRI showedwidespread confluent and focal white mattersignal change in the cerebrum and brain stem;in addition a right cerebellar-pontine angletumour was seen and thought to be a schwan-noma. At lumbar puncture CSF analysisshowed protein = 0O58 g/l, no cells were seen;stain and culture for bacteria, mycobacteria,fungi and viruses were all negative. Thepatient was thought initially to have cerebraltoxoplasmosis and began treatment withsulphadiazine and pyrimethamine. He wascounselled about the possibilities of HIVinfection; an HIV antibody test was positive.The patient was transferred to the

Middlesex Hospital for further investigationand management in October 1993. On admis-sion the signs on examination were as aboveexcept that power in the right arm was nowMRC grade 0 and in the right leg was MRCgrade 2. In addition the patient had very poorattention and was emotionally very labile.Investigations included an EEG, whichshowed focal but non-specific changes, andMRI.

MRI (Dr R Y S Chinn)This showed a mass lesion in the right cere-bello-pontine angle extending into the inter-nal auditory meatus which enhances strongly.Even in the presence of HIV infection thelesion is most likely to be schwannoma. Therewere extensive areas of high signal intensity inthe white matter of both parietal lobes on T2weighted imaging (fig 6). The abnormalitiesextend into the gyral cores without involvingthe cortical grey matter. There was no associ-ated mass effect. There were also multiple fociof high signal within the brain stem andcerebral peduncle. The regions of abnormalityhad low signal on Ti weighted imaging(fig 7). There was no enhancement after intra-venous gadolinium-DPTA. These appear-ances are consistent with progressivemultifocal leukoencephalopathy.

Dr R F MillerA repeat lumbar puncture was performed.CSF analysis showed protein = 0-6 g/l, nocells and stain and culture for bacteria,mycobacteria, fungi and viruses werenegative. Cryptococcal antigen, toxoplasmaand syphilis serology were negative in CSFand blood. DNA amplification for H simplexvirus, cytomegalovirus and varicella viruswere negative. A CD4 count was 30/mm3

It was thought on the basis of the MRIappearances and clinical presentation that thepatient had progressive multifocal leukoen-cephalopathy; therefore his anti-toxoplasmatreatment was stopped. In order to secure thediagnosis a brain biopsy was contemplated.The patient's condition rapidly deterioratedand he became increasingly drowsy, weak andincontinent. The biopsy was not performed

Figure 6 Case 2: T2 weightedMRI scan showing highsignal intensity within the white matter of both parietallobes; there is no associated mass effect.

and supportive care was given. Terminally thepatient had profuse diarrhoea and died inearly November 1993. Necropsy was per-formed.

Pathology (S B Lucas)At necropsy the patient was thin, and hadbilateral pneumonia (combined lung weight980 g) with recent right lower lobe infarction.

Figure 7 Case 2: Gadolinium enhanced Tl weightedimage showing low signal intensity within the white matterextending down into the internal capsule (arrows). There isa strongly enhancing lesion in the right cerebello-pontineangle-a schwannoma.

37

on June 4, 2020 by guest. Protected by copyright.

http://sti.bmj.com

/G

enitourin Med: first published as 10.1136/sti.71.1.35 on 1 F

ebruary 1995. Dow

nloaded from

Perrons, Chinn, Fox, Lucas, Harrison, Miller

The colon showed pseudomembranouscolitis, sparing the rectum. At the base of thebrain (weight 1490 g) was a yellow 2 cm

diameter tumour on the right eighth cranialnerve. Cortical atrophy was absent but on

slicing, ill-defined softening of the whitematter extended from the left frontal lobeback to the left parietal lobe and into bothoccipital lobes.

Histologically, the lungs had purulentbronchopneumonia and haemorrhagic infarc-tion (no emboli found). Pseudomembranouscolitis was confirmed and CMV inclusionswere not seen. The cerebral white matterlesions were PML associated with multinucle-ate giant cell encephalitis (fig 8). In the dorsalcolumns of the spinal cord there was earlyvacuolar myelopathy. The eighth nerve

tumour was confirmed as a schwannoma.

Conclusions of necropsyMajor pathologies: 1. pneumonia, 2. PML andHIV encephalitis, 3. pseudomembranouscolitis. Minor pathologies: 1. schwannoma, 2.vacuolar myelopathy.

Virology (Dr CJ Perrons and DrJ_ D Fox)CSF, peripheral venous blood and urine sam-ples taken 13 weeks (case 1) and 4 weeks(case 2) before death, and a brain tissue sampleobtained at necropsy were available from bothpatients and were screened for JC virus (JCV)DNA after amplification by the polymerasechain reaction (PCR). Approximately 2 mm3brain tissue was ground using a mortar andpestle and digested in a buffer containing SDSand proteinase K. Extracted brain tissue, CSFand urine samples were boiled and cooled onice prior to addition to the PCR mixture.Peripheral blood mononuclear cells(PBMNC) were separated from heparinisedvenous blood using Ficoll-Paque (Phar-macia); PBMNC DNA was extracted andpurified using phenol chloroform followed byethanol precipitation.

"Nested" PCR was performed using twopairs of primers specific for the T antigenregion of JCV; the second (inner) pair wasdesigned to amplify a portion of the firstround PCR product. For each sample PCRwas carried out in a Tris-based buffer contain-ing 1-5 mM MgCl1, 1-25 mM of eachdeoxynucleoside triphosphate, 1-25 units ofDNA polymerase (Ampli Taq, Perkin Elmer)and 100 ng of each primer. Ten microlitres of

Figure 8 Case 2: HIV-associated giant cellencephalitis andprogressive multifocalleukoencephalopathy.Associated with theenlarged astrocytes ofPMLare multinucleate giantcels (microglia). (x 200)(haematoxylin and eosin).

CSF, 1 ul of urine or brain extract and<2 0ug PBMNC DNA were added to thePCR mixture.

First round PCR was carried out, 4 min-utes denaturing at 94°C followed by 35 cyclesof 94°C for 1 minute [denaturing], 52°C for 1minute [annealing] and 72°C for 1 minute[extension] with an additional 7 minutesextension in the last cycle (Perkin Elmer TC 1thermocycler). After completion of the firstround PCR 1 ul of the DNA amplificationproduct was transferred to a fresh PCR mix-ture containing the "nested" inner primers.The second round of PCR (25 cycles) was

performed and these products were elec-trophoresed on agarose gel and detected byethidium bromide staining.

Detectable JCV specific DNA sequences

were identified by "nested" PCR in urine andbrain extracts from both patients and CSFfrom case 1 (table 1). Peripheral bloodmononuclear cell extracts from both patientsdid not contain detectable JCV DNA.

Discussion (ProfessorM G Harison)Both these cases show the usual clinical pre-sentation of progressive multifocal leukoen-cephalopathy (PML) with a rapidly evolvingfocal neurological deficit due to demyelina-tion of hemispheric white matter. Headache,vomiting and depressed consciousness are

rare as there is no mass effect even when thelesion affects a large part of the hemisphere.Neck stiffness, fever, and a CSF pleocytosisare rare because of the lack of a florid inflam-matory reaction in the affected area. Epilepsy isunusual as this is predominantly a white matterdisorder.' 2

There is now evidence that about 4% ofpatients with AIDS develop PML3 and thatAIDS is now the underlying cause of 55 to85% of PML. The disease was first describedin 1958 in patients who had leukaemia andlymphoma.4 Immunosuppression has been an

almost universal feature in subsequent cases;lympho-proliferative disorders have predomi-nated as underlying causes but transplantrecipients, patients on antineoplastic therapyand those with auto-immune diseases or withWhipple's disease have also been described as

having PML. Cavanagh, in 1959, suggestedthat the role ofimmunosuppression was criticaland that an opportunistic virus was thereforeprobably responsible. Virions morphologicallyidentifiable as belonging to a papova viruswere subsequently identified in the abnormalinfected oliogodendroglia of patients dyingfrom PML. The responsible virus was finallygrown from the brain of a patient with PMLwhose initials were JC.5 The JC virus, as it wasnamed, is the cause of a frequent childhoodinfection which is asymptomatic. By the ageof six years, 50% of children have serocon-

Table 1 Detection ofJCVDNA in clinical samples bynested PCR

Patient CSF Brain Urine Peripheral blood

Case 1 + + + -

Case 2 - + + -

38

-1-

.i

on June 4, 2020 by guest. Protected by copyright.

http://sti.bmj.com

/G

enitourin Med: first published as 10.1136/sti.71.1.35 on 1 F

ebruary 1995. Dow

nloaded from

Progressive multifocal leukoencephalopathy in patients with AIDS: detection ofJ7C virus DNA in CSF and brain

Table 2 Focal neurological deficit in patients with AIDS*

Feature PML Toxoplasmosis Cerebral lymphoma

Length of history weeks/months <2 weeks 2-8 weeksMotor weakness 85 50 40(mono- or hemiplegia)Speech dysfunction 30 10 40(dys- or aphasia, dysarthria)Visual deficits 30 10(homonymous hemianopiablurred vision, corticalblindness, diplopia)Altered mental state 25 60 50(personality change,dementia, impaired memory,reduced attention)Seizures 5 30 20Headache <5 55 30Depression of consciousness 1 5-30 20

*Numbers are % with specific feature: ? = unknown.

verted and by middle adulthood 80 to 90% ofthe population has been infected.6 Renaltransplant patients and pregnant women com-

monly have JC virus in the urine. It has thusbeen suggested that the kidney is the site oflatent infection. However, the genome of thevirus obtained from the urine is not always thesame as that in the brain and more recentlyHouff et a17 have shown the presence of JCvirus in the B-lymphocytes of the marrow andspleen. It has therefore been suggested thatactivation of lymphocytes in immunodeficientpatients leads to the presence of the virus inperipheral blood lymphocytes. It is then possi-ble that HIV infected macrophages in theCNS produce cytokines that are chemotactic,bringing lymphocytes through the blood brainbarrier, beginning infection of the brain withproductive multiplication ofJC virus in oligo-dendrocytes. The infected oligodendrocyte iseventually destroyed with release of virionswhich affect neighbouring oligodendrocytes;the disease therefore spreads like a bush fire.Damage to an oligodendrocyte can cause

demyelination in as little as six or seven

days.The diagnosis ofPML depends on the clin-

ical picture of focal neurological deficits with-out evidence of raised intracranial pressure on

imaging, on virological studies and ultimatelyon brain biopsy.2 Axial computed tomography(CT) brain scans, as in these cases, normallyshow low density areas within the white mat-ter with little enhancement or mass effect. Afew patients show a very faint peripheralenhancement. On MRI the lesions characteris-tically again show no mass effect and show noenhancement.8 The abnormal area is of lowsignal on T1 and on high signal on T2 showinga homogeneous appearance with a poorlydefined edge. The shape of the lesion is scal-loped because of the sparing ofU fibres at thegrey matter/white matter interface. Thespread of the lesion down the white matterpathway can often be eloquently shown on

coronal Ti imaging. The posterior fossa isaffected in 10-20% of cases; the majorityshow hemispheric white matter disease, oftenmost obvious in the parieto- occipital area. AnEEG may show non-specific slowing over thesite of focal lesions but there are no diagnosticfeatures.9 A focal EEG abnormality in a

patient with AIDS and a developing

hemiparesis, in the presence of a normal CTscan, would, however, be suspicious of PMLand prompt MR imaging should be per-formed. Routine analysis of CSF is usuallynormal, but as already discussed, detection ofJC virus-specific DNA sequences by PCR cansupport the diagnosis in the majority ofcases.The differential diagnosis of a focal

neurological deficit, in a patient with AIDS,includes cerebral toxoplasmosis which usuallyhas a shorter history, (less than two weeks), incontrast to several weeks or months withPML. While a focal deficit is commonly pre-sent with toxoplasmosis there is much moreoften an altered mental state with confusionand disorientation. Seizures are more com-mon and depression of consciousness moreusual. Headache is very common with toxo-plasmosis and rare with PML. Lymphomashould also be considered in the differentialdiagnosis with a similar history of two to eightweeks. Altered mental state, seizures anddepressed consciousness and headache areagain more likely than with PML. Cerebralinfarction, which is found at necropsy in up to25% of patients with AIDS, also produces afocal neurological deficit but this is normallyof a sudden onset and the imaging abnormalityon CT or MRI follows a vascular distribution.The imaging changes of high signal on T2

do not look like those of an enhancing toxo-plasma abscess or lymphoma but can be con-fused with local encephalitis due tocytomegalovirus or HIV: HIV encephalitis ismost unlikely to cause a hemiparesis so thecombination of the clinical picture and imagingis usually discriminatory.The prognosis after diagnosis of PML in

HIV infected patients is poor with an averagesurvival of only four months.23 Both the casesdescribed here died within that period.Occasional cases have survived a much longerperiod of time'0 and in non-AIDS patientsPML may remit if the immunodeficiencyimproves. It has therefore been suggested thataggressive antiretroviral treatment should beinstituted if at all possible. In addition cyto-sine arabinosidell and alpha interferon'2 haveboth been advocated with anecdotal accountsof individual patients showing some improve-ment. Because of the side-effect profile ofcytosine arabinoside it is usual to requirebrain biopsy confirmation of PML beforeusing it. Trials are already underway in theUSA and are planned in Europe, to seewhether the anecdotal experiences can beconfirmed in prospective studies.

1 Berger J, Kaskovitz B, Donovan Post M, Dickinson G.Progressive multifocal leuconcephalopathy associatedwith human immunodeficiency virus infection. AnnIntern Med 1987;107:78-87.

2 Sweeney BJ, Miller RF, Harrison MJG. Progressive multi-focal leukoencephalopathy. Br J Hosp Med 1993;50:187-92.

3 Sweeney BJ, Manji H, Miller RF, Harrison MJG.Progressive multifocal leukoencephalopathy at theMiddlesex Hospital 1989-91. Programme andAbstracts. Neuroscience of HIV infection: Basic andClinical Frontiers. Amsterdam 1992:137.

4 Astrom K, Mancall E, Richardson E Jr. Progressive multi-focal leucoencephalopathy. Brain 1958;81:93-1 11.

39

on June 4, 2020 by guest. Protected by copyright.

http://sti.bmj.com

/G

enitourin Med: first published as 10.1136/sti.71.1.35 on 1 F

ebruary 1995. Dow

nloaded from

Perrons, Chinn, Fox, Lucas, Hamison, Miller

5 Padgett B, Walker D, Zu Rhein G, Eckroade R.Cultivation of papova-like virus from human brain withprogressive multifocal leukoencephalopathy. Lancet1971;i:1257-60.

6 Padgett B, Walker D. Prevalence of antibodies in humansera against JC virus, an isolate from a case of progres-sive multifocal leucoencephalopathy. 7 Infect Dis 1973;127:467-70.

7 Houff S, Major E, Katz D, et al. Involvement ofJC virus-infected cells from the bone marrow and spleen in thepathogenesis of progressive multifocal leucoen-cephalopathy. NEnglJ7Med 1988;318:301-5.

8 Hansman Winterman ML, Donovan Post J, Berger JR,Tate LG, Bell MD, Limonte LP. Progressive multifocalleucoencephalopathy in 47 HIV seropositive patients;

neuroimaging with clinical and pathologic correlation.Radiology 1993;187:233-40.

9 Farrell D. The EEG of progressive multifocal leukoen-cephalopathy. Electroencephalogy Clin Neurophysiol 1969;26:200-5.

10 Berger J, Meuke L. Prolonged survival and partial recoveryin AIDS-associated leucoencephalopathy. Neurology1988;38: 1060-5.

11 Portegies P, Algra P, Hoilak C, et al. Response to cytora-bine in progressive multifocal leukoencephalopathy inAIDS. Lancet 1991;337:680-1.

12 Berger J, Pall L, McArthur J, et al. Pilot study of recombi-nant alpha 2a interferon in the treatment of AIDS-related progressive multifocal leucoencephalopathy.Neurology 1992;42(Suppl 3):257.

40

on June 4, 2020 by guest. Protected by copyright.

http://sti.bmj.com

/G

enitourin Med: first published as 10.1136/sti.71.1.35 on 1 F

ebruary 1995. Dow

nloaded from