9Journal of Neurology, Neurosurgery, and Psychiatry 1994;57:994-997

SHORT REPORT

Cortical and subcortical JC virus infection: twounusual cases ofAIDS associated progressivemultifocal leukoencephalopathy

B J Sweeney, H Manji, R F Miller, M J G Harrison, F Gray, F Scaravilli

AbstractTwo patients with AIDS and progressiveneurological syndromes had necropsiesthat identified JC virus infection ofthe cerebral or cerebeilar cortex. Theunusual presentation of progressive mul-tifocal leukoencephalopathy with greymatter involvement and normal cerebralimaging is discussed.

(J Neurol Neurosurg Psychiatry 1994;57:994-997)

Department ofNeurologyB J SweeneyH ManjiM J G HarrisonDepartment ofMedicine, UniversityCollege LondonMedical School,Middlesex Hospital,London WIP 7PN, UKR F Miller

Departement deNeuropathologie,H6pital HenriMondor, 94010Creteil, FranceF GrayDepartment ofNeuropathology,Institute of Neurology,Queen Square,London WC1N 3BG,UKF ScaravilliCorrespondence to:Dr B J Sweeney, Reta LilaWeston Institute ofNeurology, UniversityCollege London MedicalSchool, Riding HouseStreet, London W1P 7PN,UK.

Received 6 December 1993and in revised form10 February 1994.Accepted 18 February 1994

Progressive multifocal leukoencephalopathy,originally described by Astrom, Mancall, andRichardson in 1958, is an opportunistic infec-tion of the CNS by the papovavirus JC.' 2 Itbegins insidiously and is accompanied byfocal neurological signs that include sensoryand motor abnormalities, impaired vision,dysphasia, and intellectual impairment.9 Thedisease progresses rapidly and death usuallyoccurs within one year of onset.

Morphological changes include areas ofwhite-grey or yellowish discolourationlocalised to the white matter, which oftenbecome confluent.3 Within these areas myelinloss, the presence of foamy macrophages,reactive or pleomorphic astrocytes andenlarged oligodendrocytes containing ampho-philic intranuclear inclusions are the salientfeatures. The use of in situ hybridisationtechniques helps to confirm that the inclusionbodies are those of papovavirus. The myelinloss results from viral infection and subse-quent death of oligodendrocytes. The charac-teristic imaging correlates of thesepathological changes are asymmetric lesionsof white matter that do not exert a mass effector enhance with contrast.2 The issue of greymatter involvement in progressive multifocalleukoencephalopathy is discussed later.

Before the AIDS epidemic most casesoccurred in patients who had immunodefi-ciency related to neoplastic, autoimmune oriatrogenic disorders.2 The incidence hasincreased considerably since the advent ofAIDS in the early 1980s-for example, therewas a fourfold increase in the incidence ofprogressive multifocal leukoencephalopathyas a cause of death in the United Statesbetween 1979 and 1986.4 Progressive multi-focal leukoencephalopathy was first reportedin a patient with AIDS by Miller et al in 1982

and is an "AIDS-defining" illness.5 Clinicalseries have reported the incidence of progres-sive multifocal leukoencephalopathy as up to3% in patients with AIDS and as the AIDSdefining illness in up to 1%.6 Postmortemseries have given the incidence as between2-8% in patients dying of AIDS.3 Hithertothere have been no major pathological differ-ences reported between progressive multifo-cal leukoencephalopathy in AIDS andprogressive multifocal leukoencephalopathyassociated with other immunodeficiency dis-eases.7 Because of the apparently increasedincidence of progressive multifocal leukoen-cephalopathy in AIDS compared with otherimmunodeficiency states it has been sug-gested that JC virus infection may be syner-gistic with HIV infection.8 For example, it hasbeen shown that the tyrosine aminotrans-ferase protein of HIV transactivates the JCvirus in vitro.'

Whereas grey matter change associatedwith JC virus infection and progressivemultifocal leukoencephalopathy have beenreported in various clinical and pathologicalstudies, it has been as a secondary phenome-non to underlying white matter disease.6 7 10 12In this paper we report two cases of AIDSassociated progressive multifocal leukoen-cephalopathy in which JC viral infection anddestruction of grey matter and adjacent sub-cortical white matter was the major patholog-ical change. Both patients had progressiveneurological syndromes that were extensivelyinvestigated before death without a conclu-sive diagnosis being made.

Patients and methodsCASE REPORTS:Case 1 was a 32 year old man who presentedin July 1991 with a history of difficulty usinghis right hand. He was a haemophiliac whohad acquired HIV-1 infection in a bloodtransfusion given during a postoperativehaemorrhage in 1983. He had not had anyAIDS associated illnesses but had recentlycommenced prophylactic cotrimoxazole toprevent Pneumocystis carinni pneumonia. Onclinical examination there was a dyarthria,which was considered to be dystonic ratherthan cerebellar or pyramidal in origin. Therewere involuntary movements in the hands.The right hand had athetoid and myoclonic

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Cortical and subcortical JC virus infection: two unusual cases ofAIDS associated progressive multifocal leukoencephalopathy

movements. The left was clumsy and apraxic.There was a resistance to passive movementsin all four limbs that was thought to resemblegegenhalten. Attempts to sit up in bedinduced myoclonic jerks and hypertonic jerksin the legs. Myoclonic jerks were induced inthe legs by touch. Plantar responses wereflexor. There was no evidence of intellectualimpairment.

Investigations included repeated lumbarpunctures and two brain MRI scans that werenormal. Screening for toxoplasmosis andcryptococcosis was consistently negative. AnEEG was diffusely abnormal but did notshow any specific features. "Back averaging"was not performed. The myoclonus proveddifficult to control and did not respond to acombination of clonazepam, piracetam, car-bamazepine, and sodium valproate. Thepatient became progressively quadripareticand cachexic. He developed an aspirationpneumonia and died some nine months afterinitial presentation.

Case 2 was a 38 year old anti-HIV-1seropositive homosexual man who presentedin August 1990 with a four month history ofincreasing unsteadiness of gait and clumsi-ness of the right hand. He had been diag-nosed anti-HIV-1 seropositive in 1987 afteran episode of left sided herpes simplex oph-thalmicus. This was complicated by chroniccorneal infection and eventually a completeleft ptosis was induced with botulinum toxinto protect the cornea. He had pulmonarytuberculosis in 1989. Medications on admis-sion were 250 mg zidovudine twice daily and300 mg isoniazid, 10 mg pyridoxine, 50 mgdapsone daily and 50 mg fluconazole daily.

General physical examination was normal.On neurological examination he was alert andorientated. Speech was normal. He had awide based ataxic gait. Cranial nerve exami-nation showed a left ptosis and a scarred leftcornea. There was absence of light perceptionin the left eye and the left fundus could notbe visualised. There was 6/6 visual acuity inthe right eye with a full visual field, normalpupillary response, and a normal fundus.There was first degree nystagmus looking tothe right. Also, there was dysmetria on fin-ger-nose and heel-shin testing in all fourlimbs; this was more pronounced on the rightthan on the left. There was dysdiadochokine-sis in the right hand. The remainder of theexamination was normal.

Screening tests were negative for toxoplas-mosis and cryptococcosis and analysis of CSFonly showed a raised protein content. CT ofthe brain was normal and MRI showed dif-fuse atrophy but no focal lesion above orbelow the tentorium.The cerebellar syndrome progressed to the

point of making the patient confined to bed.He did not develop any other neurologicaldisorder and died three months after the ini-tial admission.

PATHOLOGICAL METHODSAfter macroscopic examination blocks frommany regions of the brains, including cerebral

hemispheres, cerebellar hemispheres, basalganglia and brainstem were processed forcelloidin (case 1) or paraffin (cases 1 and 2)embedding. Sections were stained withroutine methods.

Immunohistochemistry was performed onparaffin sections by an indirect immunoalka-line phosphatase (APAAP) method with poly-clonal antibodies raised against glial fibrillaryacidic protein (GFAP) (Dakopatts, France),Toxoplasma gondii (ICN) and herpes simplexvirus (HSV) type 1 and 2 (Dakopatts), andmonoclonal antibodies raised againstcytomegalovirus (CMV) (Biosoft, France)and varicella zoster virus (VZV) (Biosoft).HIV-Antigens were sought with monoclonalantibodies against p24 and p 17 (DuPont,UK).

In situ hybridisation was performed onparaffin embedded material after enzymaticproteolysis using buffered proteinase K at37°C with a biotylinated probe from theentire genome ofJC virus cloned onto the siteEco RI of pBR322 (Enzo Diagnostic,France) at a dilution of 1/5. DNA was dena-tured at 95°C for 30 minutes. The slides weresubsequently hybridised overnight at 42'C.Biotin was visualised with alkaline strepta-vidine-phosphatase stained by nitro bluetetrazolium/5-bromo-4-chloro-3-indolyl-phosphate.

PATHOLOGYIn case 1 macroscopic examination showedold, necrotic changes with an orange yellowdiscolouration involving the entire cortex inthe severely affected areas and limited to theU fibres in less severely affected regions.These mainly involved the precentral andpostcentral gyri and to a lesser extent thesuperior temporal gyri. Similar changes alsoinvolved the claustrum bilaterally. Theunderlying white matter, basal ganglia, brain-stem, and cerebellum were macroscopicallyunaffected. This appearance was emphasisedin a hemispheric section embedded in col-loidon and stained with the Loyez method formyelin. This showed a "moth-eaten" appear-ance of subcortical and cortical myelin withsparing of myelin in the gyri and centrumsemiovale (fig 1).At light microscopy the changes were more

extensive than they appeared on gross exami-nation and included demyelination, scantyperivascular mononuclear inflammatory infil-trates, reactive astrocytosis with large bizarreastrocytes, and numerous enlarged oligoden-drocytes with inclusions. In the macroscopi-cally affected areas, the lesions had a chroniccystic appearance with rare inclusions bearingoligodendrocytes at the periphery of necroticareas. More recent changes consisted of focaldemyelination with pronounced inflammationand numerous inclusions bearing oligoden-drocytes present throughout the U fibres,from the frontal lobes to the calcarine sulci.By contrast the underlying white matter

was normal and devoid of changes due toprogressive multifocal leukoencephalopathy,except for secondary degenerative changes

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Sweeney, Manji, Miller, Harrison, Gray, Scaravilli

Figure 1 Case 1:Coronal section of rightcerebral hemisphere at thelevel of the thalamusshowing "moth-eaten"appearance of thesubcortical and corticalmyelin (arrowed) (Loyezstain).

related to the more chronic necrotic corticalareas.

Moderate numbers of Alzheimer type IIastrocytes were present in the basal gangliabut no changes due to progressive multifocalleukoencephalopathy were seen in the stria-tum, globus pallidus, thalamus, nucleus sub-thalamicus, red nucleus, and substantia nigraof both sides. The brainstem and cerebellumwere unremarkable.

In case 2 there was no macroscopic abnor-mality visible on the hemispheres. Coronalsectioning showed normal ventricles, corticalribbon, and deep grey nuclei. The cerebellumseemed slightly shrunken and the folia werenecrotic in places.On histological examination the only

abnormalities were in the gyri of the cerebel-lum. There was a diffuse abnormality in the

Figure 2 Case 2:Photomicrograph ofcerebellar hemisphereshowing severely reducedgranular cell layer andvirtual disappearance ofmyelin. (LuxolfastbluelNissl stain; originallyx 48).

Figure 3 Case 2: Detail of cerebellar cortex showingpatchy disappearance of Purkinje cells, hyperplasia ofBergman glia, gliosis of the molecular layer, and completedisappearance ofgranular cells. (haemotoxylin-eosin stain;originally x 120).

cerebellar cortex with thinning or loss of largeareas of the granular cell layer (fig 2). Therewere reduced numbers of Purkinje cells andhypertrophy of the Bergman glia was conspic-uous (fig 3). The adjacent molecular layerhad increased cell density, predominantlycomposed of astrocytes. The white matter inabnormal folia showed vacuolation and vari-able amounts of myelin loss (fig 2). Therewere many macrophages around the smallerblood vessels. Astrocyte numbers were againincreased and many were pleomorphic.Enlarged, round nuclei with amphophilicinclusion bodies were visible throughout theabnormal areas including grey matter. A fewperivascular inflammatory cells were present.The deep cerebellar white matter, includingthe dentate nucleus, was spared.

In both cases in situ hybridisation with aprobe for papovavirus showed numerousinclusions throughout the affected white

Figure 4 Case 2: Photomicrograph illustrating thepresence ofJC virus by the in situ hybridisation method.(originally x 300).

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Cortical and subcortical JC virus infection: two unusual cases ofAIDS associated progressive multifocal leukoencephalopathy

matter and also through all the layers of over-lying grey matter (fig 4). Staining for p24antigen was negative. There were no otherfocal lesions, micro-organisms were notdetected on sections stained or immuno-stained for viruses, bacteria, fungi, or para-sites. There were no multinucleated giantcells and immunostaining for HIV was nega-tive.

DiscussionThese cases are the first to record an associa-tion between a progressive neurological disor-der and predominant involvement of the greymatter and immediately subcortical structureswith JC virus infection without evidence ofwhite matter disease on MRI. Case 1 pre-sented with involuntary movements-namely,myoclonus and athetosis, and then developedprogressive quadriparesis. Case 2 had a pro-gressive cerebellar syndrome.

There have been previous reports onpathological and clinical evidence suggestingthat progressive multifocal leukoencephalopa-thy can cause abnormalities in grey matter.Richardson in 1961 and more recentlyKuchelmeister and colleagues have describedabnormal cells in the granular cell layer in thecerebellum of patients with adjacent whitematter progressive multifocal leukoen-cephalopathy with and without AIDS.10 "These cells had nuclei twice the size of nor-mal granular cell nuclei and had little cyto-plasm. Kuchelmeister et al" did not find anylocal granular cell layer infection with JCvirus in these cases. These authors did notfind a reduced number of granular cells. Wedid not see these cells in our cases.

Clinical reports of patients with progressivemultifocal leukoencephalopathy developingseizures and extrapyramidal disorders haveconsidered grey matter involvement althoughwithout gross grey matter disease at post-mortem.6 Hansman Whiteman et al haverecently reported an abnormal signal fromdeep cerebral grey matter on MRI of patientswith AIDS and progressive multifocalleukoencephalopathy. 12 Pathologically thesechanges were related to progressive multifocalleukoencephalopathy in adjacent white matterand to destruction of myelinated fibres pass-ing through the basal ganglia structures. Aswe have already indicated MRI was negativein our patients.

In a histological study Aksamit et al foundJC virus by in situ hybridisation in satelliteoligodendrocytes in grey matter overlyingwhite matter foci in four of 10 patients withAIDS associated progressive multifocalleukoencephalopathy and in glial cells in thegranular cell layer of the cerebellum in twopatients with progressive multifocal leukoen-cephalopathy not associated with AIDS.7

These findings were not associated withobvious histopathological abnormality as inthe cases described here.To the clinician these cases represent a

hitherto unrecognised presentation of JCvirus infection. The classic features of whitematter involvement without mass effect orcontrast enhancement on CT or MRI wereabsent and thus there was no evidence fromprevious knowledge to suggest progressivemultifocal leukoencephalopathy as a diagno-sis.2 This may question the use of the term"leukoencephalopathy" which implies whitematter involvement as an essential feature ofthis disease. Perhaps adoption of the term"JC virus encephalitis" would prevent thismisunderstanding in the future.

In conclusion, we highlight the unusualfeatures of these cases of progressive multifo-cal leukoencephalopathy: predominant infec-tion of the cortex and adjacent white matterand lack of imaging abnormality. Both of thepatients had AIDS. This pattern of clinicaland pathological involvement has to ourknowledge not previously been reported inprogressive multifocal leukoencephalopathynot associated with HIV-1.

BJS and HM are funded by the Medical Research Council ofGreat Britain. We would like to thank Dr B De Toffol foraccess to the clinical description of case 1.

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3 Scaravilli F, Gray F, Mikol J, Sinclair E. Progressivemultifocal leukoencephalopathy. In: Scaravilli F, ed. Theneuropathology ofHIV infection. Berlin: Springer Verlag,1993:113-7.

4 Holman RC, Janssen R, Buehler JW, Zelasky BS, HooperWC. Epidemiology of progressive multifocal leuko-encephalopathy in the United States: analysis ofnational mortality and AIDS surveillance data.Neurology 1991;41: 1733-6.

5 Miller JR, Barrett RE, Britton CB, et al. Progressive multi-focal leukoencephalopathy in a male homosexual withT-cell immune deficiency. N Engl J Med 1982;307:1436-8.

6 Berger JR, Kaskovitz B, Donovan Post MJ, Dickinson G.Progressive multifocal leukoencephalopathy associatedwith human immunodeficiency virus infection. AnnIntern Med 1987;107:78-87.

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8 Vazeux R, Cumont M, Girard PM, et al. Severeencephalitis resulting from co-infections with HIV andJC virus. Neurology 1990;40:944-48.

9 Tada H, Rappaport J, Lashgari M, Amini S, Wong-StallF, Khalili K. Trans-activation of the JC virus latepromoter by the TAT protein of type 1 humanimmunodeficiency virus in glial cells. Proc Natl Acad SciUSA 1990;87:3479-83.

10 Richardson EP Jr. Progressive multifocal leukoen-cephalopathy. NEnglJ7Med 1961;265:815-823.

11 Kuchelmeister K, Bergmann M, Gullotta F. Cellularchanges in the cerebellar granular layer in AIDS-associ-ated PML. Neuropathol Appl Neurobiol 1993;19:398-401.

12 Hansman Whiteman ML, Donovan Post MJ, Berger JR,Tate LG, Bell MD, Limonte LP. Progressive multifocalleukoencephalopathy in 47 HIV-seropositive patients:neuroimaging with clinical and pathological correlation.Radiology 1993;187:233-40.

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