histological, immunofluorescent, ultrastructural features ... · histological, immunofluorescent,...

Br J Vener Dis 1984;60:390-5

Histological, immunofluorescent, and ultrastructuralfeatures of lymphogranuloma venereum:A case report

B ALACOQUE,* H CLOPPET,t C DUMONTEL,* AND G MOULIN*From the *Department of Dermatovenereology, Hopital de l'Antiquaille, Lyon, the fDepartment ofImmunochemistry, Institut Pasteur, Lyon, and the *Department of Histology, A Carrel MedicalUniversity, Lyon, France

SUMMARY We studied the course of a case of lymphogranuloma venereum (LGV) over twoyears. The comparative histological, immunological, and ultrastructural studies showed theexistence of characteristic granulations within the macrophages of the granuloma.We suggest that direct immunofluorescence is a specific method for diagnosing cutaneous LGV.

Some ultrastructural aspects lead us to believe that different chlamydial bodies exist inside thegranuloma.

Introduction

To diagnose lymphogranuloma venereum (LGV) anumber of criteria are necessary: characteristicclinical symptoms, compatible histology, andpositive results to serological tests for chlamydial(LGV serotypes) antibodies. Electron microscopycan be a useful adjunct to diagnosis, but is notroutinely performed. The most specific diagnostictest is culture of the organism from material obtainedfrom the patient. If culture is negative, diagnosis canonly be presumptive. We have tried to provide aspecific test based on direct immunofluorescence oftissue samples. Our ultrastructural study providedevidence of atypical chlamydial bodies during theirlife cycle in LGV.

Patient and methods

A 22 year old Italian women was admitted to hospitalin May 1980 for investigation of condylomatous peri-neal lesions that had developed two years previously.There was no relevant history; she had not travelledoutside Europe, and had not had extramarital sexualintercourse. The condition had started in April 1978during the second month of her first pregnancy,when three or four painful, purulent, punctiformpapules developed. The lesions were found on the

Address for reprints: Dr B Alacoque, Department of Dermato-venereology, Hopital de l'Antiquaille, I Rue de l'Antiquaille, 69321Lyon cedex 1, France

Accepted for publication 12 March 1984

external and inferior aspects of the left labiummajus, which was oedematous, erythematous, andpainful but not indurated. There were no systemicsymptoms. Because the lesions spread to the wholevulvar area her baby was delivered by cesarean sectionin October 1978. The course of the pregnancy hadbeen normal, and the neonate was normal. After thebirth the lesions became hypertrophic and condy-lomatous and spread to the perineo-anal region.There were no urinary or gastrointestinal symptoms,but she had dyspareunia. No diagnosis was made.Different medical treatments were ineffective. Thepatient consulted us in May 1980.Examination showed vulvoperineal esthiomene

(fig 1). The lesions were composed of large condy-lomatous formations (2 cm in diameter), erythe-matous papules (I mm in diameter) disseminatedover healthy and condylomatous skin, and papularvesicles resulting from ectasia of the lymphaticvessels. On the vulva the condylomata were on theexternal aspect of the labia majora; the vaginalorifice and labia minora were unaffected but some-what oedematous. Smaller identical lesions werefound around the gluteal fold and the anal margin.General and proctological examination showed noabnormalities, and the vagina was normal. Treatmentwith sulphonamides, oxytetracycline, and erythro-mycin produced resolution of the dyspareunia, butlittle improvement in the condylomatous lesions.

LABORATORY FINDINGSResults of complement fixation tests for LGV were<1/8, and of microimmunofluorescence tests <1/40.

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Histological, immunofluorescent, and ultrastructuralfeatures of lymphogranuloma venereum

Urethral cultures failed to yield chlamydiae. Biopsyspecimens were not cultured. Other bacteriological,parasitological, and virological cultures and sero-logical tests gave negative results.

INVESTIGATIONSBipedal lymphography was normal. Kinmonth'smethod with blue patent violet showed a quick anderratic impregnation of the ectasia of the lymphaticvessels.

Several biopsies of the vulvar esthiomene were per-formed using the following stains and methods:haematoxylin, eosin, and safran; May-Grunwald-Giemsa, immunoperoxidase followed by May-Grunwald-Giemsa; direct immunofluorescenceimmunoperoxidase; and electron microscopy.

ImmunofluorescenceThe fluorescent antibody technique was performedon deparaffinised 5 Mm thick sections.' Antiserum toChlamydia trachomatis was obtained either from apatient with chlamydial urethritis (test dilution 1/10)or from hyperimmune rabbits (test dilution 1/100)kindly supplied by Dr Thouvenot, Faculty ofMedecin, A Carrel Medical University, Lyon.Fluorescent antiserum was respectively goatantihuman IgG (Institut Pasteur, Paris, France)diluted 1/100 and antirabbit IgG (Institut Pasteur)diluted 1/100. The specificity of the tests wascontrolled by a fluorescence inhibition procedure byprevious incubation of some sections withheterologous antichlamydial reagent. The specificitywas also controlled by incubation withimmunofluorescent conjugate after omission of thehuman and rabbit immune serum respectively. Alldilutions were performed in phosphate bufferedsaline (PBS) ph 7 - 2.

Immunoperoxidase procedureThe immunocytological reaction was performed oneither paraffin sections or semithin sections. Serialsections of 5 urm paraffin sections were mounted onglass slides with aqueous gelatin, deparaffinised intoluene, and rehydrated. Semithin (1 um) sectionswere obtained from the samples used for the ultra-structural study. They were mounted on gelatin pre-coated glass slides. To remove Araldite, the sectionswere exposed to ethanolic sodium hydroxide diluted1/1 with 100% ethanol for 10 minutes. After threewashes in 100%o ethanol, the sections wererehydrated and then bleached for 10 minutes in0 03%o hydrogen peroxide.The sections were stained by an indirect immuno-

'peroxidase method. They were incubated overnightwith the patient's immune serum diluted 1/10 to 1/80in trometamol (TRIS) buffered saline (ph 7-6),

followed by incubation with sheep antihuman IgGconjugated with peroxidase (Institut Pasteur) diluted1/100. The peroxidase substrate was hydrogenperoxide 3-3' diamino-benzidine. The sections weremounted with Entellan (E Merck, Darmstad, WestGermany). This study was controlled by theincubation of sections with sheep antihuman IgGlabelled with peroxidase after omitting the humanimmune serum or substituting for it a non-immunehuman serum at the same dilution.

Electron microscopyPieces of condylomatous lesions were removed andcut into small pieces. They were fixed separately in40o glutaraldehyde and 207o formalin buffered withcacodylate 0 32 mol/l (ph 7 - 4). They were post-fixedin 2% osmium tetroxide buffered at ph 7X4. Afterbeing washed, tissues were dehydrated in gradedethanol and embedded in Araldite. Ultrathin sectionsprepared with a Reichert OMU 3 were contrastedwith uranyl acetate and lead citrate. They wereexamined and photographed in a Jeol JEM 7 electronmicroscope.

Results

HISTOLOGYIn some areas sections stained with haematoxylin,eosin, and safran showed acanthosis of the epidermiswith noticeable papillomatosis; in other areas theepidermis was almost normal. The papillary dermiswas oedematous and had dilated lymphatic vessels.The reticular dermis presented a very dense fibrousappearance. It contained inflammatory cell infiltrates,which were often in clusters throughout the dermisbut were most frequently located around the vessels.The inflammatory granuloma was polymorphous,with lymphocytes, plasma cells, and sometimesmultinucleated giant cells. The infiltrates were foundas far down as the deep dermis close to the sweatglands. Free pigment was present in the dermis.

Sections stained with May-Grunwald-Giemsashowed features generally similar to those describedabove. Many mast cells were seen disseminated insidethe granuloma. Their cytoplasm was full of small,spherical, blue granulations. These granulations weresometimes disseminated in the tissue or formedclusters far from or next to the mast cells. Theiracidophilic colour was more prominent outside thecell. In addition to the numerous mast cells, a fewmacrophages were seen. They were located near thedilated lymphatic vessels either alone or in clusters oftwo to 10 cells in the vicinity of the inflammatorycells. The cytoplasm of these macrophages was filledwith large granulations. They were 0 * 4 pm to 3 *2 pmin size, irregularly shaped, and in contrast to the

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Histological, immunofluorescent, and ultrastructural features of lymphogranuloma venereum

FIG 5 Macrophage with large atypical inclusions (x 12 600) (FIB =fibroblast, MAC macrophage. Insert:Double unit membrane limiting the inclusions - (x 65 000).

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azurophil staining of the mast cells, were heavilystained dark blue by May-Grunwald-Giemsa. Theseintracytoplasmic granulations stained with May-Grunwald-Giemsa could be "Gamma Bodies"2 firstdescribed by Favre3 then Gamma.4 Mast cell andmacrophage granulations were more easily distin-guished by staining with May-Grunwald-Giemsaafter immunoperoxidase. Similar granulations werealso seen in the intercellular spaces (fig 2).

DIRECT IMMUNOFLUORESCENT ANTIBODYSTUDIESAt low magnification (x 250), despite slight back-ground fluorescence of the whole sections, fluorescentelements were seen clearly in the granulomatousareas. The number of fluorescent elements variedfrom one field to another in a given section and fromone section to another in a given sample. Thesefluorescent elements occurred either alone or inclusters of two to 10. At higher magnification(x 400) the fluorescence was made up of smaller,irregularly shaped, clustered elements inside the cells.The cell nuclei were not fluorescent. In thegranuloma fluorescent elements were also found out-side the cells. Their specific fluorescence suggestedtheir chlamydial nature (fig 3).

IMMUNOPEROXIDASE STAININGIn paraffin sections at low magnification (x 250)brown cells could easily be seen inside the granuloma.They predominated at the level of the nodular infil-trates. At higher magnification (x 400) the labellingwas clearly inside the cytoplasm of macrophages.Pigment was found in clusters or was distributed out-side the cells. The specific labelling of these elementssuggested that they were chlamydial (fig 4). Nolabelling was noted in stained semithin sections.

ELECTRON MICROSCOPY STUDIESIn semithin sections (stained methylene blue azure II)the reticular dermal infiltrate was moderately denseand mainly composed of macrophages and lympho-cytes. There were also some monocytes, neutrophilgranulocytes, and plasma cells. Macrophages withlarge granulations were characteristic. Capillariesdisplayed a dilated cleft shape.Only macrophages were seen in thin sections

(counterstained with uranyle acetate and lead citrate)(fig 5). They were irregularly shaped and had manypseudopods. The Golgi apparatus was well developed.Mitochondria were numerous, and there were manylysosomes especially secondary lysosomes.

In most cells, different types of inclusions werefound within the cytoplasm. Some were spherical(0 3 to 0*35 ,Mm in diameter), always included in avacuole, and had one or several eccentric nucleoids.

B Alacoque, H Cloppet, C Dumontel, and G Moulin

These structures had ultrastructural features identicalto those of elementary bodies (fig 6). Other inclusionswere spherical (0- 5 Mm in diameter) with one or

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FIG 6 Elementary body with macrophage and osmiophilinclusion (Ce = elementary body, V= vacuole) a(x 3500).

several nucleoids in the centre of the structure. Thesestructures were compatible with intermediate bodies.Further inclusions were very large (up to 2 or 3 jAm)and were scattered throughout the cytoplasm. Theywere limited by a double unit membrane. Theygenerally had a homogeneously dense electron con-tent, although lamellar structures were occasionallyseen. At the periphery of the cytoplasm a number ofsmall nucleoids could be found. These inclusionswere difficult to identify (fig 5, insert). Initial bodieswere rare. Only one binary fission was observed (fig7). In two or three cells we noted one large vacuolelimited by a single unit membrane containing somestructures that had undergone necrosis.

Discussion

Our investigation concerned a case of LGVdiagnosed two years after the onset of symptoms.Although the patient had received antibiotics beforeconsulting us, in our opinion the diagnosis of LGVwas not in doubt. Indeed our positive findings con-firmed this diagnosis whereas the negative results didnot refute it, and it was supported by the clinical andhistological findings. Positive specific serology (1/40)was atypical but this could be related to previoustreatment. As is well recognised in the advancedstages of LGV, the condylomatous lesions hardly

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Histological, immunofluorescent, and ultrastructural features of lymphogranuloma venereum

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responded to treatment. Urethral culture for Ctrachomatis gave negative results, but for technicalreasons it could not be performed on granulomatoustissue. This negative result does not exclude thediagnosis, however, and other diagnoses are unlikely.In 1904 Favre3 and Gamma4 found granulationswithin macrophages in histogical sections stainedwith May-Grunwald Giemsa and suggested that theycould be all or part of the then unknown agentcausing the disease. In the opinion of Sheldon andHeyman they were more likely to be the result ofphagocytosis and not significant.5

In our study similar granulations were found onstaining with May-Grunwald-Giemsa, immuno-fluorescence, immunoperoxidase, and on electronmicroscopy. Their topography inside the granuloma,intramacrophage site, size, shape, and staining werelike those of the structures described in 1924.3 4 Thespecificity of the immunological reactions confirmedthat they were chlamydiae. Immunofluorescencespecificity was checked by using two antibody andconjugate systems in parallel and by specificextinction studies. The failure of labelling byimmunoperoxidase after removing Araldite fromsemithin sections can be explained by ethanolicsodium hydroxide having denatured antigenic sites.On electron microscopy, however, there were not

many intracytoplasmic inclusions displaying theclassic ultrastructural features of chlamydiae thatwere described in culture by Eb et al.6 They do notaccount for the intense immunofluorescent andimmunoperoxidase reactions. It may be that not onlythe classic chlamydial inclusions but also the atypicalintracytoplasmic inclusions are both chlamydialbodies. Although the ultrastructure of the latter may

be modified by the granulomatous reaction, anti-biotic treatment, or both, these atypical inclusionsmay retain their antigenicity.A study of the action.of penicillin on Chiamydia

psittaci showed that it has an effect on the binaryfission of initial bodies, causes abnormal forms toappear, and modifies the ultrastructural aspect of theelementary body wall.7 Philipp and Metz studied theultrastructural aspects of C trachomatis isolatedfrom a patient with LGV during the initial stage ofthe disease and before treatment and noted thenormal cycle of development of chlamydiae withtheir characteristic structures.8 In our study tissuewas removed for examination two years after theonset of clinical signs and after a number of anti-biotic treatment regimens, which may explain theatypical features found.As no similar study existed with which to compare

our results on the immunocytology or ultrastructureof LGV, we wish merely to report our observationsso that a number of hypotheses may be confirmed orrefuted by further studies. Should these hypothesesbe confirmed, a specific diagnosis of LGV could bemade based mainly on the immunocytological studyof biopsy specimens with well characterised anti-chlamydial antibodies; monoclonal antibodies wouldmake this diagnosis more reliable and standardised.More accurate histological and ultrastructuralknowledge of the cycle of chlamydiae in the granu-lomatous reaction would also be possible.We suggest that direct immunofluorescence is a

simple, rapid, and specific method for diagnosingLGV. Moreover, the advantage of this method is thatit allows retrospective diagnosis on previous biopsies.

We thank Dr Y Gille of the bacteriology department,Hopital de l'Antiquaille, Lyon and Dr Y Li of the histologydepartment, Alexis Carrel Medical University, Lyon fortheir technical help.

References

1. Sainte Marie G. A paraffin embedding technique for studiesemploying immtinofluorescence. J Histochem Cytochem1962; 10:250.

2. Colimon R, Morel P. Maladie de Nicolas et Favre. In:Maladies infectieuses. Paris: Encyclopedie Medico-Chirurgicale, 1979 (8076 A-10).

3. Favre M. Sur la lymphogranulomatose inguinale subaigue. LaPresse Medicale 1924; 62:651-2.

4. Gamma C. Sur l'etiologie de la lymphogranulomatoseinguinale subaigue. La Presse Medicale 1924;37:404-5.

5. Sheldon WH, Heyman A. Lymphogranuloma venerum. Ahistologic study of the primary lesion, bubonulus, and lymphnodes in cases proved by isolation of the virus. Ant J Pathol1947; 23:653-65.

6. Eb F, Devauchelle G, Orfila J. Etude ultrastructurale del'agent de la lymphogranulomatose venerienne. Journal deMicroscopie 1972; 13:47-56.

7. Matsumoto A, Mantre GP. Electron microscopic observationson the effects of penicillin on the morphology of Chlamydiapsittaci. J Bacteriol 1970; 101: 278-85.

8. Philipp N, Metz J. Erregernachweiss bei lympho-granulomatosis inguinalis. Eine elecktron mikroskopisheundersuchung. Hautarzt 1975;26:41 1-5.

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