GENITAL INFECTION IN ASSOCIATION WITH TRIC VIRUS

Brit. J. vener. Dis. (1964), 40, 25. GENITAL INFECTION IN ASSOCIATION WITH TRIC VIRUS INFECTION OF THE EYE II. CYTOLOGY.* PRELIMINARY REPORT BY M. KHALAF AL-HUSSAINI AND BARRIE R. JONES The Institute of Ophthlmology, London The im of this investigtion ws to study the cellulr chnges occurring in TRIC virus infection of the genitl trct, nd to compre them with the chnges occurring in infection of the eye by this gent. * Red t the Meeting of the M.S.S.V.D. in Copenhgen, June 7, 1963., i. K*I R L L ii IC i I LI 1. I TIU I.1I d'c1i'ick'-icl nci Ȯ'iT uo LI IC 11 I C LI 11 - ~~~~~~~~ ~CF 'l It 1 ;>t 1 tltc 1 l11.if I 111I 11 T11\ t' ;1 AND ERIC M. C. DUNLOP Whitechpel Clinic, The London Hospitl _t.. Method Scrpings of the epithelium of urethr nd cervix were tken gently (Dunlop, Jones nd Al-Hussini, 1964). Only gentle scrping is required becuse the inclusion bodies occur in the superficil lyers of the epithelium, s cn be seen in the section of the conjunctiv shown in Fig. 1....,...,.4.. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~........ Br J Vener Dis: first published s 10.1136/sti.40.1.25 on 1 Mrch 1964. Downloded from http://sti.bmj.com/ Adk: MOfEF 2 Alb on 22 September 2018 by guest. Protected by

26 BRITISH JOURNAL OF VENEREAL DISEASES After smers hd been mde on slides nd dried, they were fixed in methyl lcohol, nd stined by Giems's method. Mcchivello's nd Cstfied's stins, whilst stisfctory for demonstrting inclusion bodies, do not revel the cellulr pttern. Iodine stining (Rice, 1936; Gilkes, Smith, nd Sow, 1958), which is invluble for the rpid demonstrtion of inclusions in conjunctivl scrpings (Fig. 2), cnnot be used on genitl mteril, becuse mny cells, in the urethrl nd cervicl epithelium contin glycogen nd therefore stin so intensely with iodine (Jones, Collier, nd Smith, 1959) tht the recognition of inclusions is impossible (Fig. 2b). 9. FIG. 2.-() Conjunctivl scrpings stined with iodine, showing crbohydrte mtrix of inclusion body (x 1,000). (b) Cervicl scrpings stined with iodine, showing stining of cytoplsm of epithelil cells ( x 1,000). TYPES OF CELL FOUND (A) Epithelil Cells (1) Norml Epithelil Cells.-Norml urethrl epithelil cells (Fig. 3b) resemble conjunctivl epithelil cells (Fig. 3) very closely. Ech hs round nucleus 10 to 12 VL in dimeter. The purple-stining chromtin hs regulr, finely grnulr pttern. The cytoplsm is fintly blue nd slightly grnulr in ppernce; the cell is usully between 20 nd 25,u in dimeter. Two types of norml epithelil cells re commonly seen in cervicl scrpings: trnsitionl cells (Fig. 3c), which re very similr to conjunctivl epithelil cells, nd squmous epithelil cells which my become kertinized (Fig. 3d). O.-I-- FIG. 3.-Norml epithelil cells (Giems x 1,000). () Conjunctiv. (b) Urethr. (c) Cervix, trnsitionl cells. (d) Cervix, kertinized squmous cells..:.. r ': Br J Vener Dis: first published s 10.1136/sti.40.1.25 on 1 Mrch 1964. Downloded from http://sti.bmj.com/ on 22 September 2018 by guest. Protected by

_ st. R:.:: S.. N ' X =.. GENITAL INFECTION WITH TRIC VIRUS INFECTION OF THE EYE- II 27 The ltter re lrge cells with smll, dense blue swollen nuclei re left bre. In the erly stges, the nuclei which become more purple s they ge; the nucler chromtin loses its uniformly fine grnulr bundnt cytoplsm my be ple blue or colourless ppernce. Its ffinity for dyes lessens so tht it nd often contins some purple kertin grnules; ppers blurred nd ple. Spces develop between the virus inclusion bodies re not found in these cells ple chromtin msses nd enlrge to form multiple (Thygeson nd Stone, 1942). vcuoles or rmifying clefts. The nucleoli lso become swollen nd pler. The enlrged nucleus with its (2) Abnorml Epithelil Cells.-As result of locl expnded chromtin pttern often elongtes nd TRIC virus infection, mny of the epithelil cells of becomes indented or kidney-shped, so tht the cell the cervix, urethr, nd conjunctiv show chrcteristic toxic or degenertive chnges (Fig. 4). The cells s some of them re phgocytic. Epithelil cells, cn my esily be confused with monocyte, especilly become enlrged up to 30,u in dimeter. The nucleus however, be identified by the presence of nucleoli. becomes swollen, commonly reching 15 [ in These progressive chnges, with loss of cytoplsm, dimeter, but the cytoplsm becomes vcuolted s led to the ppernce of mny ple, swollen, nd the process continues, nd then disintegrtes so tht blurred nucler ghosts. b b -I s r F... g:./ \.w... ^ *s,t,.+,. -i,e.g c. q.n.v X.: s _ffi... _l i_ - w. -_ I FIG. 4.-Epithelil cells showing degenertive chnges (Giems x 1,000). FIG. 5.-Phgocytic cells (Giems x 1,000). () Conjunctiv. (b) Urethr. (c) Cervix. () Conjunctiv. (b) Urethr. (c) Cervix. Br J Vener Dis: first published s 10.1136/sti.40.1.25 on 1 Mrch 1964. Downloded from http://sti.bmj.com/ on 22 September 2018 by guest. Protected by

28 BRITISH JOURNAL OF VENEREAL DISEASES (3) Phgocytic Epithelil Cells.-Phgocytic cells similr to the Leber cells of trchom (Duke-Elder, 1938) re found in most of the scrpings from both urethr nd cervix when these sites re infected by TRIC virus (Fig. 5, previous pge). Their nucler pttern nd the presence of nucleoli frequently indicte n epithelil origin. (4) Epithelil Cells with Inclusions.-The developmentl cycle of Hlberstedter-Prowzek inclusion *.X.Se.i :.::... bodies (Hlberstedter nd Prowzek, 1907) strts - with the entry of n elementry body (O * 25 to O 5 [L in dimeter) into n epithelil cell. The prticle swells to become reltively lrge blue-stining initil body (0 3 to I1 5,u in dimeter) which then divides successively to form cluster of smller initil bodies (Fig. 6). This cluster is referred to s round bsophil inclusion body (Fig. 6b). As the inclusion enlrges, incresing numbers of purplestining elementry bodies pper within it (Fig. 6c). ~~~~~~~---- ---I-i---;---... FIG. 6.-Stges in development of TRIC virus inclusion bodies (Giems x 1,000). () Initil bodies (rrow) nd smll bsophil inclusion body in (d) Crescentic inclusion bodies forming cps to the nuclei the djcent cell (conjunctiv of Bby D). (conjunctiv). (b) Multiple bsophil inclusions; two smll round inclusions, one (e) Mture eosinophil inclusion body (conjunctiv). c-hpdinclusionboyf irgundesnting (thee.nucleus, nd one lrger (f) Ruptured epithelil cell liberting free elementry bodies (c) Inclusion body in urethrl cell from Mr C (ffow). Br J Vener Dis: first published s 10.1136/sti.40.1.25 on 1 Mrch 1964. Downloded from http://sti.bmj.com/ on 22 September 2018 by guest. Protected by

0...,*,...... GENITAL INFECTION WITH TRIC VIRUS INFECTION OF THE EYE- II 29 At this stge, the inclusion body is often crescentshped nd forms cp on the nucleus (Fig. 6d). The drk-ground illumintion revels striking golden exmintion of the Giems-stined scrpings by mture inclusion (Fig. 6e) fills the cytoplsm nd fluorescence of the virus prticles within the inclusion body (Figs 7c nd 8b). ruptures, liberting free elementry bodies (Fig. 6f) to repet the cycle in other cells. In differentiting inclusions from other structures it should be relized tht more thn one inclusion body my be found in single cell (Figs. 6b nd 7). In conjunctivl scrpings, the demonstrtion by iodine-stining of glycogen in the mtrix of the L.. inclusion leves no doubt bout its nture (Fig. 7). This is not prcticble with genitl scrpings, but :..::~ ~ ~ B- "-# *.pr-......i5 :: :.s :.:... sfie *-; *' R A. AIML :M..i *:.-._ *_ - FIG. 7.-Conjunctivl epithelil cell from Mr L, with two Hlberstedter-Prowzek inclusion bodies (x 1,000). () Stined with iodine. (b) Sme field decolourized nd stined with Giems. (c) Sme field stined with Giems s seen with drk-ground illumintion. FIG. 8.-Cervicl epithelil cell from Mrs D showing n inclusion body (Giems x 1.000). () Ordinry illumintion. (b) Drk-ground illumintion A nucler protrusion (Fig. 9, overlef) cn be identified becuse the colour nd pttern of the extruded mteril re identicl with those of nucler chromtin. Furthermore, brek in the nucler membrne cn sometimes be seen. Occsionlly deep blue-stining grnultion of the cytoplsm occurs (Fig. 9b). This my ssume shpe resembling tht of n inclusion body, but the remining cytoplsm often shows similr grnultion. This "psuedo-inclusion" is pure blue, unlike initil bodies which re nvy blue nd elementry bodies which re purple; furthermore, it is not composed of the shrply-defined prticles which constitute the virus inclusion. The cytoplsm of cervicl epithelil cells sometimes hs pink grnultion which is probbly due to kertin (Fig. 9c). The grnules, which re scttered throughout the cytoplsm, re irregulr in shpe nd size nd re often lrger thn elementry bodies. Bcteril ggregtes (Fig. 9d) sometimes occur in or on the cytoplsm of urethrl or cervicl cells. The Br J Vener Dis: first published s 10.1136/sti.40.1.25 on 1 Mrch 1964. Downloded from http://sti.bmj.com/ on 22 September 2018 by guest. Protected by

30 Discussion In TRIC virus infection of the eye, the dignostic fetures in descending order of sensitivity re: (1) Chrcteristic clinicl findings. (2) Chrcteristic cellulr chnges in scrpings. (3) Presence of inclusions. (4) Isoltion of virus. The precise significnce of positive complementfixtion test with the psittcosis-lymphogrnulom- TRIC virus (PLT) group ntigen nd tht of intrderml tests remin to be ssessed in reltion to TRIC virus infections (Dunlop nd others, 1964). Clinicl exmintion by n experienced observer will frequently estblish n unequivocl dignosis of one or other of the TRIC virus syndromes. Sometimes the findings re equivocl. Cellulr chnges consistent with clinicl dignosis of TRIC virus infection, but without virus inclusions, mke the dignosis likely but do not prove it. The finding of inclusions or the isoltion of virus leves no doubt, but this proof becomes incresingly difficult to obtin with the pssge of time fter infection. For ll the ptients presenting with eye disese, with the exception of one who hd lredy received tretment (Mrs 0), n unequivocl dignosis of oculr TRIC virus infection ws estblished by finding inclusions in conjunctivl scrpings. In conjunctivl scrpings the finding of mny epithelil cells which show the typicl sequence of "toxic" or degenertive chnges, together with mixed inflmmtory exudte of polymorphs, lymphocytes, occsionl plsm cells, lrge monoi d b.c. C... FiG. 9.-Pseudo-inclusions (Giems x 1,000). () Nucler protrusion (conjunctiv). (b) Grnulr cytoplsm (conjunctiv). (c) Kertin grnules (cervix). (d) Bcteril ggregte (urethr). BRITISH JOURNAL OF VENEREAL DISEASES IRMW IEL individul prticles re lrger in size nd more bluestining thn those of virus inclusions. Similr ggregtes my be found outside the cell nd they do not fluoresce like TRIC virus elementry bodies when exmined by drk-ground illumintion. (B) Inflmmtory Cells Neutrophil polymorphonucler leucocytes re constntly present in the inflmmtory response to TRIC virus infection of the eye. They re the predominnt cells in the erly stges nd continue to be found so long s virus persists. They were present lso in ll the genitl scrpings in which the chrcteristic degenertive chnges were recognized in epithelil cells. An occsionl eosinophil my be seen in these scrpings from both eye nd genitl trct. Lymphocytes re numerous, plsm cells nd lrge primitive mononucler cells re less commonly seen. As noted bove, phgocytic cells occur in these scrpings (Fig. 5b nd c); they usully pper to be epithelil cells, rther thn monocytes or tissue mcrophges. Br J Vener Dis: first published s 10.1136/sti.40.1.25 on 1 Mrch 1964. Downloded from http://sti.bmj.com/ on 22 September 2018 by guest. Protected by

GENITAL INFECTION WITH TRIC VIRUS INFECTION OF THE EYE-H nuclers, nd phgocytic cells, is comptible with dignosis of TRIC virus infection. If the chnges re pronounced, they suggest this dignosis. It seems likely tht these findings hve similr significnce when observed in genitl scrpings. However, the only cytologicl proof of TRIC virus infection is the finding of Hlberstedter-Prowzek inclusion bodies. It my resonbly be ssumed tht the mothers (Mrs D, E, I, C, H) of the five bbies suffering from neontl TRIC virus ophthlmi hd TRIC virus infection of the genitl trct, sufficiently ctive to infect the bbies t birth. In ll five cses (Tble I), the cellulr chnges in the cervicl scrpings were consistent with dignosis of TRIC virus cervicitis; in one (Mrs D) inclusions were found in the scrpings nd virus (IOL-4/GCx) ws isolted from them (Jones, Al-Hussini, nd Dunlop, 1964). Similrly, the cellulr chnges in the urethrl scrpings were consistent with dignosis of TRIC virus urethritis in ll three of the fthers exmined; inclusions were found in urethrl scrpings from ll of them nd virus (IOL-9/GU) ws isolted from the urethr of Mr H (Tble I). Hd these prents been exmined erlier in the course of their genitl infections, it is likely tht inclusions would hve been found (Lindner, 1910) nd virus isolted from higher proportion. In the cse of eight dult ptients suffering from TRIC virus infection of the eye nd four of their consorts, no inclusions were found in genitl scrpings, nor ws virus isolted from them (Tble II). However, cellulr chnges consistent with the dignosis of TRIC virus infection of the genitl trct were present in nine of the ptients tested. These chnges, the clinicl findings, nd the serologicl results (Dunlop nd others, 1964) suggest tht genitl TRIC virus infection ws present in some cses t lest. Further work in progress confirms this ssocition. Although, under idel conditions, the finding of inclusions is esier thn isoltion of virus, it is still procedure tht is prcticble only in reserch study. It demnds the expenditure of gret del of time by trined observer; even then, filure to find inclusions does not exclude the dignosis. If indequtely-trined observers re employed, if hurried exmintions re mde, or if the method of collection of mteril is indequte, the results will lmost certinly be negtive. We re investigting nd compring the reltive sensitivity of immunofluorescent techniques. Although there re considerble difficulties, it is possible tht fluorescentntibody method my ultimtely provide the best dignostic procedure for TRIC virus infection of the genitl trct (Nichols, McComb, Hddd, nd Murry, 1963). TABLE I CYTOLOGICAL FINDINGS IN GENITAL SCRAPINGS FROM EIGHT PARENTS OF FIVE BABIES WITH TRIC VIRUS OPHTHALMIA NEONATORUM Cytology No. Comptible No. No. Prents of with with of Virus Prents TRIC Virus Inclusions Isoltes Infection Mothers.5 5 (D, E, 1, C, H) (Mrs D) (Mrs D) Fthers.3 3 3 1 (D, C, H) (Mr D, C, H) (Mr H) TABLE II CYTOLOGICAL FINDINGS IN GENITAL SCRAPINGS FROM EIGHT ADULT PATIENTS WITH TRIC VIRUS EYE INFECTION AND FOUR OF THEIR CONSORTS Cytology Oculr Number Comptible No. with No. of Syndrome of with Inclusions Virus Cses TRIC Virus Isoltes Infection Inclusion Conjunctivitis 3 ptients+ 2 consorts 3 0 0 Punctte Kerto-conjunctivitis 2 ptients+ I consort 3 0 0 Trchom.3 ptients± I consort 3 0 0 Totl.12 9 0 0 3 1 Br J Vener Dis: first published s 10.1136/sti.40.1.25 on 1 Mrch 1964. Downloded from http://sti.bmj.com/ on 22 September 2018 by guest. Protected by

32 BRITISH JOURNAL OF VENEREAL DISEASES Summry The findings in scrpings from the genitl trct ffected by TRIC virus re described nd compred with those in scrpings from the conjunctiv in TRIC virus infection of the eye. The chnges were identicl. The methods for demonstrting TRIC virus inclusions re discussed nd the differentil dignosis of these inclusions is considered. A typicl picture of degenertive cellulr chnges nd inflmmtory exudte ws found in cervicl scrpings from the mothers of five bbies suffering from TRIC virus neontl conjunctivitis. In one mother TRIC virus ws isolted from cervicl scrpings nd inclusions were present. The sme cellulr chnges were present in urethrl scrpings from the three fthers who were exmined; ll were found to hve "non-specific" urethritis. Inclusions were present in the urethrl scrpings obtined from ll three nd TRIC virus ws isolted from one. Scrpings from the genitl trct were lso exmined in the cse of eight dults suffering from TRIC virus infection of the eye, nd four of their consorts. The chrcteristic cellulr chnges suggesting TRIC virus infection were found in nine of the twelve subjects; lthough inclusions were not found nd virus ws not isolted, it seems likely tht t lest some of these ptients hd genitl infection with TRIC virus. REFERENCES Duke-Elder, S. (1938). "Text-book of Ophthlmology", vol. 3, p. 1519. Kimpton, London. Dunlop, E. M. C., Jones, B. R., nd Al-Hussini, M. K. (1964). Brit. J. vener. Dis., 40, 33. Gilkes, M. J., Smith C. H., nd Sow, J. (1958). Brit. J. Ophthl., 42, 473. Hlberstedter, L., nd von Prowzek, S. (1907). Arb. Gesundh.-Amte (Berl.), 26, 44. Jones, B. R., Al-Hussini, M. K., nd Dunlop, E. M. C. (1964). Brit. J. vener. Dis., 40, 19. Collier, L. H., nd Smith, C. H. (1959). Lncet, 1, 902. Lindner, K. (1910). Wien. klin. Wschr., 23, 283. Nichols, R. L., McComb, 0. E., Hddd, N., nd Murry, E. S. (1963). Amer. J. trop. Med., 12, 233. Rice, C. E. (1936). Amer. J. Ophthl., 19, 1. Thygeson, P., nd Stone, W. (1942). Arch. Ophthl. (Chicgo), 27, 91. Infection genitle vec infection oculire due u virus TRIC 1I. Cytologie REsuME On decrit les frottis genitux de mldes infectes pr le virus TRIC et on les compre ux frottis conjonctivux de mldes tteints d'infection oculire pr le virus TRIC. On discute l demonstrtion des inclusions du virus TRIC et le dignostic differentiel. On trouv une degenertion cellulire exudt inflmmtoire dns les frottis du col de l'uterus chez les meres de 5 enfnts tteints de conjonctivite neontle due u virus TRIC. On isol ce virus des frottis du col de l'uterus d'une des meres, et on y trouv des inclusions. L meme degenertion fut presente dns les frottis uretrux de 3 peres exmines; tous etient tteints d'uretrite non-specifique. On trouv des inclusions dns les frottis de tous les trois et on isol le virus TRIC d'un pere. On exmin les frottis genitux de 8 dultes tteints de virus TRIC oculire et de 4 de leurs prtenires sexuels. On trouv l degenertion clssique de l'infection pr le virus TRIC dns 9 cs sur 12; on ne trouv ps d'inclusions et on n'isol ps de virus, mis il se peut que quelques mldes fussent tteints d'infection genitle due u virus TRIC. Br J Vener Dis: first published s 10.1136/sti.40.1.25 on 1 Mrch 1964. Downloded from http://sti.bmj.com/ on 22 September 2018 by guest. Protected by