J Clin Pathol: first published as 10.1136/jcp.30.9.847 on 1 September 1977. Downloaded from J. clin. Path., 1977, 30, 847-850 Direct immunofluorescence in the diagnosis of toxoplasmic lymphadenitis ROBERT M. MATOSSIAN', VICTOR H. NASSAR2, AND AHMED BASMADJI2 From the Department ofBacteriology and Virology', and the Department ofPathology2, American University ofBeirut, Beirut, Lebanon SUMMARY The diagnosis of toxoplasmic lymphadenitis was established through the demonstration, by direct immunofluorescence, of toxoplasma cysts and trophozoites in a cervical lymph node biopsy which also had a characteristic histopathology. The patient had cervical lymphadenopathy and increased specific fluorescent antitoxoplasma IgG and IgM antibodies. The inclusion of Toxoplasma gondii infections among Case history the syndromes responsible for human cervical lymphadenopathy has necessitated the use of specific A 24-year-old Palestinian sailor was seen at the to differentiate this condition from a American of procedures University Beirut with a history of a copyright. more serious illness (Putschar, 1973). In such gradually increasing number of palpable nodules in patients the presence of a raised titre of antibodies, the neck of one year's duration. Physical examination detected by the Sabin-Feldman dye test, has usually of the cervical area revealed enlarged lymph nodes suggested recent exposure to the protozoon (World of the occipital and submaxillary regions. These Health Organisation, 1969). The co-existence of were firm, discrete, and non-adherent to the skin or either complement-fixing (CF) or IgM antibodies, deeper tissues. A lymph node biopsy was obtained obtained by indirect immunofluorescence (IF), has from the occipital region and diagnosed as being often been correlated with acute infections (Reming- suggestive of toxoplasmic lymphadenitis. Blood http://jcp.bmj.com/ ton et al., 1968; Dorfman and Remington, 1973). samples were subsequently sent for diagnostic The histopathology of lymph nodes, characterised by serology. follicular hyperplasia, associated with clusters of epithelioid histiocytes, is considered to be charac- PATHOLOGY teristic of toxoplasma lymphadenitis (Gray et al., The specimen consisted of a single formalin fixed 1972; Dorfman and Remington, 1973). T. gondii has occipital lymph node (1x2 cm diameter) with no also been isolated from lymph nodes injected into gross lesions. Histopathological examination revealed laboratory mice. However, individual Toxoplasma, reactive follicular hyperplasia where the follicles on September 30, 2021 by guest. Protected or cysts containing clusters of trophozoites, have exhibited unusual variation in size and shape. rarely been observed in fixed stained histological Within the enlarged germinal centres, macrophages preparations from lymph nodes. This has often showing active phagocytosis were present. In handicapped the pathologist in his interpretation of addition, there was the distinctive presence of focal preparations where the histopathology is not typical. clusters of pale staining epithelioid histiocytes, We report the results of a study in a patient with which were noted within the interfollicular lymphoid cervical lymphadenopathy who had increased titres stroma encroaching upon the germinal centres. of specific fluorescent IgG and IgM antibodies to Occasionally, the sinuses appeared packed with T. gondii, where a diagnosis was achieved by the smaller histiocytes. The lymph node stroma con- characteristic histopathology of the lymph node and tained plasma cells and large immunoblasts (Fig. 1). the demonstration by direct immunofluorescence of Toxoplasma cysts and trophozoites within the IMMUNOFLUORESCENT STUDIES lymphoid tissues. Indirect fluorescent antibody (IFA) test Received for publication 3 February 1977 Circulating IgG and IgM antibodies were demon- 847 J Clin Pathol: first published as 10.1136/jcp.30.9.847 on 1 September 1977. Downloaded from 848 Robert M. Matossian, Victor H. Nassar, and Ahmed Basmadji copyright. Fig. 1 Posterior cervical lymph node. There is remarkable follicular hyperplasia in which the macrophages exhibit phagocytosis ofnuclear matter. There is also distinctive aggregation of epithelioid histiocytes in irregular clusters (granulomatoid) which are scattered in the interfollicular stroma and encroach upon the germinal centres. Haematoxylin and eosin x 30. http://jcp.bmj.com/ strated by using T. gondii freeze-dried trophozoite the lymph node was observed. The follicular areas antigens and monospecific anti-human IgG and IgM were faintly fluorescent with patchy areas of more conjugates'. The procedure employed has been intense staining. Under higher magnification these previously described by Kane et al. (1971). The serum were made up of clusters of what appeared to be anti-toxoplasma IgG and IgM titres were 1/1000 and trophozoite-like bodies, surrounded by a non- 1/100, respectively. fluorescent membrane (Fig. 2). The lymph sinusoids were with fluorescent bodies, similar to congested on September 30, 2021 by guest. Protected Direct immunofluorescence those seen in the clusters. The cells filling the Thin sections from the paraffin embedded lymph sinuses were engorged with fluorescent material. A node were passed through xylene and fixed on slides large number of free-lying intensely staining, single, with methanol. The area to be tested was flooded crescent-shaped bodies were also seen. When with a 1 in 10 dilution of the anti-toxoplasma compared with a preparation of known T. gondii conjugate'. The slides were incubated in a humid freeze-dried trophozoites, the fluorescent bodies seen chamber for 30 minutes. They were then rinsed in in the biopsy specimen had an appearance consistent phosphate buffered saline (PBS), pH 7x2, for 30 with T. gondii. Similar ininunofluorescent studies on minutes. The slides were next dried and mounted five lymph node biopsies from Hodgkin's lymphoma, with coverslips using 10% glycerol in carbonated tuberculous lymphadenitis, sarcoidosis, and non- phosphate buffer. They were examined by the specific lymphadenitis were all negative. conventional method for immunofluorescence (Car- Histopathological observation of lymph node ver and Goldman, 1959). biopsies are considered highly suggestive if not A diffuse fluorescence of the capsular structure of characteristic of toxoplasma lymphadenitis (Gray et al., 1972). The distinctive histopathologica 'Obtained from Wellcome Reagents Ltd, England features are a triad: (1) Striking follicular hyper- J Clin Pathol: first published as 10.1136/jcp.30.9.847 on 1 September 1977. Downloaded from Direct immunofluorescence in the diagnosis oftoxoplasmic lymphadenitis 849 Fig. 2 Section of cervical lymph node, stained with anti-toxoplasma conjugate, demonstrating fluorescent trophozoite-like bodies (arrows) surrounded by a non-fluorescent membrane. x 750. copyright. plasia; macrophages within the germinal centres similar observation was made by Tsunematsu et al. exhibiting phagocytosis of nuclear particles, im- (1964) in two patients with toxoplasmic lymphadeno- parting a 'starry-sky' appearance to the enlarged pathy. germinal centres, and marked mitotic activity. (2) Toxoplasma may persist in lymph nodes, skeletal Distinctive aggregation of pale staining epithelioid muscle, and other tissues for months or years after histiocytes in irregular clusters (granulomatoid) infection. Their presence may thus be unrelated to scattered in the inter-follicular stroma, often en- the active disease. However, the detection of the http://jcp.bmj.com/ croaching upon the follicles. (3) Packing of the proliferative forms of Toxoplasma in miscellaneous dilated sinusoids, both peripheral and central, with tissues should be of help in establishing an active smaller monocytoid cells. Plasma cells and immuno- infection. Hence their presence in patients with blasts are present in the lymphoid stroma. However, definite lymphadenopathy may be sufficient evidence the above classical pattern may represent a narrow to make the diagnosis. range within a broader spectrum. At either end of We suggest the use of direct immunofluorescence the spectrum the specific histological picture may as a sensitive and specific test in the diagnosis of on September 30, 2021 by guest. Protected not obtain. active toxoplasmic lymphadenitis. We present this The direct staining of T. gondii cysts and free- communication, hoping that others will have a lying trophozoites with fluorescein-labelled antibody better opportunity further to assess the diagnostic has been achieved in sections of spleen, liver, lung, value of this procedure. and brain of mice experimentally infected (Carver and Goldman, 1959). Fluorescent Toxoplasma have We thank the Wellcome Research Laboratories for also been observed in the retina of laboratory their free supply of Toxoplasma gondii freeze-dried infected rabbits (Tabbara et al., 1974). Archer et al. antigen, anti-human IgG and IgM conjugate, as (1971) demonstrated the specificity and sensitivity well as anti-toxoplasma conjugate. of direct immunofluorescence in the diagnosis of ovine abortion. Our data indicate that crescent- References shaped bodies, single or in clusters, forming cysts Archer, J. F., Beverley, J. K. A., and Watson, W. A. fluoresce with specific anti-toxoplasma conjugate (1971). A field trial of the fluorescence antibody test for in thin sections of lymph node obtained from toxoplasmosis in the diagnosis of ovine abortion. formalin fixed and paraffin-embedded tissue. A Veterinary Record,