further observations in urinary sediment in - jcp.bmj.com · varicella herpeszoster measles...

J. clin Path. (1967), 20, 835

Further observations on inclusion-bearing cellsin urinary sediment in infectious diseases

J. F. BOYD AND NADA NEDELKOSKA1

From the Brownlee Laboratory, the University Department of Infectious Diseases,Ruchill Hospital, Glasgow, N. W., and the University Department ofPathology,

Western Infirmary, Glasgow, W.2

SYNOPSIS A study of the cytology of the urinary sediment in 43 patients with known viral diseaseshas revealed a variety of inclusion-bearing cells in 28.The morphology of the cells suggest that the changes recorded may be due to the viral infec-

tions, at least in some instances, bearing in mind the findings of workers quoted in our 1964 reportthat cellular changes very similar to those induced by virus infections can be initiated by non-viralstimuli. Multinucleate giant cells are occasionally found in chickenpox, measles, herpes simplexinfection, and in mumps.

The work of Bolande (1959 and 1961) stimulated usto examine the cytology of urinary sediment, andsome of our preliminary observations have beenrecorded (Boyd and Nedelkoska, 1963 and 1964).These showed that inclusion-bearing cells werenoted in 25 of 100 patients consecutively admittedto Ruchill Hospital, the 'positive' cases includingfive with varicella, two with herpes zoster, the othercases including central nervous system, respiratory,alimentary, and cutaneous conditions for which noviral aetiology was proved. Among the 75 'negative'cases, there were patients with illnesses of acceptedviral aetiology, for example, rubella, primary herpessimplex, mumps, infective hepatitis, homologousserum jaundice, poliomyelitis (type 3 strain), andenteritis associated with ECHO virus type 2 infec-tion. Although consideration ought to be given tothe study of urinary sediment from normal (i.e.non-infected) children and adults, this investi-gation has not been pursued in view of the difficultyof defining normality. Appreciation of the existenceof latent virus infections and of prolonged excretionof virus in the urine after some infections might makethis line of pursuit less appropriate.Our next approach therefore has been to examine

a consecutive series of admissions with illnesses ofaccepted viral aetiology, and the results are pre-sented in this article.

'British Council scholar. Present Address: The Clinic for InfectiousDiseases, Skopje, Yugoslavia.Received for publication 6 April 1967.

MATERIAL AND METHODS

A fresh specimen of urine was obtained from the patienton the first morning after admission to hospital and wastreated in the same fashion as described in our earlierreports (Boyd and Nedelkoska, 1963 and 1964). Sincethese specimens were obtained usually about 7 a.m. andwere processed between 10 and 11 a.m., the routine wasestablished that the specimens were stored in Universalcontainers (30 ml., 1 fl. oz.) at ward temperature ratherthan in a refrigerator. The heavy precipitation of uratesand phosphates which tended to occur if the lattermethod was adopted seriously obscured the study ofcells. Specimens which showed heavy bacterial growthduring this period were discarded as being unsatisfactory;those showing only a few bacteria were retained. None ofthe patients included in this report belongs to the earlierseries.The criteria for 'positive' and 'negative' cells and for

'positive' and 'negative' urine samples are unchanged.

RESULTS

The results of the investigation are shown in theTable. Of 43 patients with illnesses of known viralaetiology, 28 (65%) showed inclusion-bearing cellsin the urine in varying numbers. This result differsvery greatly from that of the earlier series. Ourwider experience has shown that there is great varia-tion in the cellularity of urine specimens even in aseries of patients with the same viral illness, and wenow appreciate that some patients exhibit abacterialpyuria during the course of virus illnesses, an

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TABLEINCIDENCE OF POSITIVE CASES SH(

BEARING CELLS IN URINARY SDiagnosis

VaricellaHerpes zosterMeaslesHerpes simplexPoliomyelitisRubellaMumps

Total

observation which has also been nCherry (1965).There are 13 males and 11 fema

their ages ranging from 3 months15 are positive. Only one of the n

showed no exfoliated cells in theThis group fails to support the coresults of our earlier series.Two more patients with herpes z

and 73-year-old females, are repoiurine specimen was positive. Cytoweak positive result. This findingtrast to the two examples quotereports. The discrepancy may becondition remaining localizedexamples. One of the earlier exlocalized, however, but the urine s

theless moderately strongly positiveThe most persistently positive re

in measles (morbilli), where fiNpositive of six examined (five mEwith the sixth case yielding an aceladmission.There are three patients who E

have primary herpes simplex infboys, aged 61 yr. and 1 yr., andThe girl and the older boy yieldelesions and showed good antibodyounger boy, whose urine was acelland was therefore classified as n

yield herpes simplex virus fromserology was not performed.The three patients with poliomy

31 yr. and 5 yr., and a boy of 1

urinary specimens on admissioicellular one, from the boy, wasclusion-bearing cells. The other cpositive. The infection was due toin all instances.Of the three rubella patients, tI

men was from a 2-year-old girlurine sample was acellular. One opatients, a 21-year-old boy, had

J. F. Boyd and Nada Nedelkoska

from hospital three weeks earlier after recoveryOWING INCLUSION- from chickenpox, and it is uncertain therefore'EDIMENT whether or not his urinary inclusion-bearing cellsNo. of Positive were all due to rubella: it is possible that the recentPatients Cases chickenpox infection contributed partly to this24 15 result.2 1 One of the patients with mumps, not seen during3 2 the acute stage, was admitted with epididymo-3 2 orchitis, and his urine was negative. Serology for2 1 mumps infection was not performed. The other

patient, an 18-year-old girl, was a typical case of43 28 mumps, both clinically and serologically. Her

admission urine specimen was positive even althoughnade by Hart and it was not very cellular.

The present series has given us further experienceles with varicella, with the study of inclusion-bearing cells in urinary

; to 37 years, and sediment and the descriptions given in ourmine negative cases earlier series hold good for the present one. Theurinary sediment. most commonly affected cell is mononuclear, butnsistently positive some are binucleate, and examples of multinucleate

giant cells have been encountered in chickenpox,zoster, 64-year-old measles, herpes simplex, and in mumps as describedrted and only one below. The colours which are detailed relate to thelogically it was a original Papanicolaou-stained preparations and notis a marked con- to the black and white illustrations printed here.ed in our earlier Figures 1 and 2 show cells encountered in onedue partly to the case of varicella. The first cell has the size and somein the present of the features of a urinary tract transitional cell.iamples was also In the rather ballooned nucleus there is a smallample was never- spherical deeply blue-black body. Near the peripherya. of the nucleus there is a smaller grey sphere, tenta-sults are obtained tively labelled a 'polar body', and between these twove patients were structures there is a delta-shaped green intranuclearales, one female), inclusion. The nucleoplasm is pale green. Thellular specimen on position of the nuclear membrane is noticeable and

is green. The cytoplasm is pale green and possessesare considered to numerous tiny red inclusions of apparently equalections. Two are size. The second cell possesses four ballooned nucleia girl of 3 years. although only three are in focus. Two show red,d virus from the intranuclear structures of rather irregular shape andly responses. The in close relationship to blue-black bodies, one oflular on admission which is larger than the others and is ovoid. Theegative, failed to staining reactions are similar to the cell of Figure 1his lesions and and this cell also shows numerous tiny red cyto-

plasmic bodies of apparently equal size. This multi-relitis, two girls of nucleate giant cell is quite unlike the variety normallyyr., gave cellular encountered in smears of vesicle fluid in chickenpox.n and the least Our studies of measles show that mononuclearnegative for in- and binucleate inclusion-bearing cells are encoun-,ases were weakly tered in the urine, thus confirming the observationspolio-virus type 1 of Bolande, and that in addition, multinucleate

giant cells, similar to the syncytial forms met withhe negative speci- in the giant-cell pneumonia of measles, may bewhose admission noted occasionally, thus confirming observationstf the two positive made by Bolande (1959) and by Mitus, Enders,been discharged Edsall, and Holloway (1965). Figures 3 and 4 show



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Further observations on inclusion-bearing cells in urinary sediment in infectious diseases

.o'w:... ~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~! . ......

Flu;. b FIG. 7

FIGS. 6 and 7. H. simplex. Papanicolaou. x 1,300.

FIG. 1 FIG. 2

FIGS. 1 and 2. Chickenpox. Papanicolaou. x 1,600.

.~';

FIG. 8. Poliomyelitis. Papanicolaou. x 1,300.

FIG. 3

FIG. 9 FIG. 1 0

FIGS. 9 and 10. Rubella. Papanicolaou. x 1,300.

FIG. 4

FIGS. 3 and4. Measles. Papanicolaou. x 1,600.

FIG. 5. H. simplex. Papanicolaou. x 1,300.FIG. 1 1 FIG. 12

FIGS. 11 and 12. Mumps. Papanicolaou. x 1,300.

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two examples from the same patient. Figure 3 showsa large cell with four nuclei, all being virtually infocus. In each instance the nuclear chromatin formsa condensed blue-black solid sphere, and a dull redmoderately enlarged spherical structure, consideredto be the nucleolus, is present alongside. Within thesurrounding homogeneously orange nucleoplasmthere are a few small red and blue-black satellitestructures not all of which are distinguishable in theillustration. It is uncertain if the position of thenuclear membrane is stained or whether the inter-face is the result of contrast staining reactions alone.The cytoplasm is moderately green and vacuolatedperipherally, but it is red and granular centrally, andthere is a biconcave spherical red inclusion whichbears specks of unidentified refractile material at oneor two points. The multinucleate giant cell ofFig. 4 possesses seven nuclei although only one isclearly in focus. Only two have been shown clearlyto possess bodies which could be enlarged nucleoli;one is dull red, and the other is grey or very paleblue. The chromatin of each nucleus is condensed asa blue-black bar, sphere, or ovoid, and the nucleo-plasm is clear and unstained in each instance. As inthe case of the cell in Fig. 3, the limits of a nuclearmembrane are well defined but whether or not theweak eosinophilia is genuine, or is a refractory arte-fact, is uncertain. The cytoplasm is red and granularand carries a red crescentic inclusion.

Figures 5, 6, and 7 are taken from a patient withherpes simplex infection and show different features.The cells in Fig. 5 bear a strong resemblance tourinary tract epithelial cells. They show somewhatballooned nuclei, neither of which shows a nucleolus.Both nuclei show the blue-black chromatin to becondensed on the nuclear membrane as biconvexmasses of almost equal size. In one instance, thedistribution is moderately even, resembling a rosary,whereas in the other case it is disinctly uneven, andlarge gaps, resembling enlarged nuclear annuli, are

visible. Centrally, the one nucleus shows a fainthomogeneously stellate grey inclusion with clearincomplete halo, whereas the other nucleus shows a

rather grey and white marbled nucleoplasm. Thecytoplasm of one cell bears eosinophilic granuleswhich are aggregated into masses of different sizeand shape, and the cytoplasm is pink centrally andgreen peripherally while the cytoplasm of the othercell is moderately green and bears occasionalvacuoles.

Figure 6 shows a different situation. This mono-

nuclear cell shows ballooning of the nucleus whichis a feature of herpes simplex virus infection.Centrally, there is a deep red body, believed to be an

enlarged nucleolus with a small red polar bodyalongside in clear nucleoplasm. The blue-black

chromatin is condensed as large biconvex masses ona thin nuclear membrane on which there are alsonumerous very small, faintly staining biconvexmasses. The cytoplasm is moderately green andvacuolated, and possesses a large orange cytoplasmicinclusion which is out of focus. There are severalsimilar smaller inclusions on the cytoplasmic mem-brane. This particular cell shows some resemblanceto cells from the urine sediment of a patient withgeneralized zoster infection and illustrated in Figs.3, 4, and 6 of Boyd and Nedelkoska (1964), butnone of these cells show a red nucleolus or a red'polar body'. The cell illustrated in Fig. 7 possessesthree nuclei, but only two are in focus. All nucleiare ballooned. Two show no convincing nucleoliwhereas the third (the smaller shown in Fig. 7)shows an unstained structure which might benucleolus with a surrounding blue-black layer. Twonuclei show blue-black biconvex chromatin massesof varying sizes on a thickened nuclear membrane,whereas the third nucleus (which is out of focus)only shows a thickened nuclear membrane. Thenucleoplasm of the nuclei shows areas of rarefaction.The cytoplasm is moderately green and vacuolatedand shows many red inclusions some of which arearranged in a crescentic row.

Figure 8 shows a mononuclear cell from a patientwith poliomyelitis and this cell shows a blue-blackcondensed sphere of chromatin in green homo-geneous nucleoplasm. There are two red polar bodies,one or both of which may be nucleoli. Only one isin focus. While the position of the nuclear membraneis identifiable it is doubted if any staining is present.The cytoplasm is moderately green centrally, wherethere is a red paranuclear inclusion, and it is palerperipherally where there are several unstainedvacuoles.

Mononuclear and binuclear inclusion-bearingcells have also been noted in rubella. Figure 9 showsone variety of binucleate cell. In each instance, thenucleus appears to possess an unstained sphericalnucleolus, and the blue-black chromatin is condensedaround this structure as a sphere in one case and asa cigar-shaped body in the other. The nucleoplasmis green, and the position of the nuclear membraneis shown although no nuclear membrane is visible.Scanty small blue-black biconvex masses, however,are present along the perimeter here and there. Thecytoplasm is green and an orange spherical inclusionis present although it is out of focus. Figure 10 isanother binucleate cell showing essentially similarfeatures. The nucleoli are unstained. The nucleo-plasm is green. The cytoplasm is pale green andgranular and bears three moderate-sized red in-clusions and many small ones.

Figure 11 shows a multinucleate giant cell in the

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Further observations on inclusion-bearing cells in urinary sediment in infectious diseases

urinary sediment of a patient with mumps. Allthree nuclei are ballooned and possess nucleoli whichare vermilion red while two nucleoli also possessblue-black margins. The nuclear chromatin isblue-black or grey and stippled, and also formssmall biconvex masses of varying size on thickenedblue-black nuclear membranes. The nucleoplasm isclear and grey. The central nucleus shows the sexchromatin (or Barr body) against the nuclear mem-brane. The cytoplasm is grey, green and granular,with vacuolated areas, and showing small pinheadgrey inclusions below the central nucleus. Thebinucleate cell in Fig. 12 shows unstained nucleoliwith condensed blue-black nuclear chromatin. Thesurrounding nucleoplasm is deep green. No distinctnuclear membrane separates it from the grey-orangegranular cytoplasm which is vacuolated and bears amoderate-sized homogeneous orange inclusion andseveral smaller ones.

DISCUSSION

In general, the present study shows that there aremany varieties of fundamental change which canaffect cells in the urinary sediment. We cannot statecategorically that the changes recorded in anyparticular cell are the consequence of infection bythe virus responsible for the patient's disease,although we consider that the results as a wholecould be given such an interpretation.

Bolande (1959) noted inclusion-bearing cells infour urine specimens of five studied from patientswith varicella but his description of the cell type is ageneral one and makes no reference to cells of thetype shown in Figures 1 and 2. In their study of thecytopathology of parainfluenza type 3 virus infectionin HeLa and monkey kidney cells in vitro, however,Love and Suskind (1961) show intranuclearinclusions of similar appearance to those shown inFigures 1 and 2.

In prodromal measles, multinucleate giant cellformations are encountered regularly in the lym-phoid tissue throughout the body as the Warthin-Finkeldey giant cell, and occasionally giant cells, arefound in the lungs when pneumonia occurs. Thegiant cells in the former situation bear hundreds ofpyknotic nuclei usually placed centrally in the cellwhich seldom shows cytoplasmic inclusions, whilethe syncytial forms in the lung alveoli classicallyshow both intranuclear and cytoplasmic eosino-philic inclusions. The cells which are illustrated inFigures 3 and 4 resemble closely the syncytial giantcells even although eosinophilic intranuclear in-clusions are absent. Sherman and Ruckle (1958)found a bladder mucosal giant cell in a patient withmeasles, although their illustration is not very

convincing. Giant cells of the variety illustratedhave not been noted in all patients with measles,however.The descriptions of cells infected with herpes

simplex virus mention that there is inhibition ofmitosis, that the nuclei are ballooned, have losttheir nucleoli, show margination of the nuclearchromatin on the nuclear membrane, and that theremay be a large central inclusion which is usuallyeosinophilic and possesses a halo. It is clear, how-ever, that this description, which applies both to themononuclear as well as to the multinucleate giantcell, is only one instant in the course of a virus cyclein an infected cell. Love and Wildy (1963) havestudied the ribonucleoproteins of HeLa cellsinfected with herpes simplex virus and have detailedmany changes during the course of infection. Onecell in Fig. 5 may show a classical A inclusion whilethe other cell may show A granules appearing withinthe A inclusion and, alongside, the two solitaryblue-black spheres may be 'B' bodies, as describedby these authors. The large red 'nucleolus' in Fig. 6may represent the pars amorpha of the nucleolus asdescribed by early histologists, a term which hasbeen employed effectively by Love in several pub-lications in order to distinguish it from the nucleoliniof the nucleolus, which appear to be invisible in thecell illustrated. The cell in Fig. 7 shows areas of rare-faction in the nucleoplasm, also described byLove andWildy. It is debatable whether or not the smallernucleus could be classified as a micronucleus,another feature recorded by Love and Wildy (1963)and more recently by Boiron, Tanzer, Thomas, andHampe (1966) in tissue culture experiments.As mentioned earlier, it was our impression that

the urine samples from patients with poliomyelitiswere not as strongly positive as, for example, werethose from patients with measles. The cell illustratedin Fig. 8, although fixed and stained differently fromthe method employed on tissue cultures by Reissig,Howes, and Melnick (1956), appears to be similarto type 6 of their classification of progressive cyto-logical changes after infection with poliovirus.Cowdry type B inclusions (1934) were not noted inthis series, and this may be explained by the findingof Reissig et al. that type B inclusions are presentabout four hours after infection and are found intype 2 cells of their classification, that is to say,about halfway in the course of the viruscycle through the cell. Hart and Cherry (1965)noted inclusion-bearing cells in the urine of childrenafter oral poliovirus vaccine and were rather un-certain about the significance of this finding.Abnormal urinary cytology was not found bySchultz and Neva (1966) in their experimentalstudies of poliovirus infections of mice and rats.

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One characteristic of earlier work on rubellagrown on tissue cultures was the absence of anynoticeable cytopathic effect, although more recentwork (e.g., Veronelli and Maassab, 1965) appears toshow that cytopathic effects can be produced if asuitable cell line is chosen. The above authorsdescribe eosinophilic cytoplasmic inclusions in theirexperiments. Bolande (1959) reported finding in-clusion-bearing cells in the urine of three patientswith rubella out of four cases studied. Further,Heggie and Robbins (1964) reported the presence ofinclusion-bearing cells in seven of 15 urine specimensfrom naval recruits with rubella while only one of14 specimens from control subjects showed in-clusion-bearing cells. We would not place anysignificance on the apparent difference between theincidence of positive results achieved by these authorsand by ourselves. Our positive samples were notvery strongly positive by our arbitrary criteria.Utz and Szwed (1962) cultured mumps virus

from 58 out of 110 urine specimens from 21 patients.Bolande (1959) reported inclusion-bearing cells inthe urine of two patients with mumps and that thespecimens were moderately positive. A solitarypatient with mumps and submandibular abscessfailed to reveal these cells in our previous publication

(Boyd and Nedelkoska, 1964) and only one of thetwo patients in this series was positive. We havebeen unable to find any evidence that other workershave noted multinucleate giant cells similar to thatshown in Fig. 11 in the urine sediment of suchpatients.

We wish to thank W. Marshall F.I.M.L.T. for histechnical assistance.

REFERENCES

Boiron, M., Tanzer, J., Thomas, M., and Hampe A. (1966). Nature(Lond.), 209, 737.

Bolande, R. P. (1959). Pediatrics, 24, 7.-(1961). New Engl. J. Med., 265, 919.Boyd, J. F., and Nedelkoska, N. (1963). Scot. med. J., 8,269.-,- (1964). J. Path. Bact., 88, 115.Cowdry, E. V. (1934). Arch. Path., 18, 527.Hart, A. F., and Cherry, J. D. (1965). New Engi. J. Med., 272, 174.Heggie, A. D., and Robbins, F. C. (1964). Ibid., 271, 231.Love, R., and Suskind, R. G. (1961). Exp. Cell Res., 24, 521.-, and Wildy, P. (1963). J. Cell Biol., 17, 237.Mitus, A., Enders, J. F., Edsall, G., and Holloway, A. (1965). Arch.

ges. Virusforsch., 16, 331.Reissig, M., Howes, D. W., and Melnick, J. L. (1956). J. exp. Med.,

104,289.Schultz, I., and Neva, F. A. (1966). J. Immunol., 96, 74.Sherman, F. E., and Ruckle, G. (1958). Arch. Path., 65, 587.Utz, J. P., and Szwed, C. F. (1962). Proc. Soc. exp. Biol. (N.Y.),

110,841.Veronelli, J. A., and Maassab, H. F. (1965). Arch. ges. Virusforsch.,

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