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since cilia would have little to do with carcinogenesis in the colon. A thorough review of the literature disclosed no report of any patient with cancer as well as PCD. PCD itself is fairly uncommon, the incidence of which in Sweden is about 1:20,000.8 In the English literature, only seven cases of situs inversus and concomitant malignant tumor have been reported.5 The rarity, however, does not preclude the importance of identifying the congenital condition, particularly in the event of surgical treatment. Special attention was directed to postoperative respiratory management of our patient. Our effort to clarify the variety of symptoms led to detection of the congenital syndrome and subsequently successful treatment of the carcinoma.

References I. Afzelius BA: A human syndrome caused by immotile cilia. Science 1976; 193:317-319

Afzelius BA: "Immotile cilia" syndrome and ciliary abnormalities induced by infection and injury. Am Rev Respir Dis 1981; 124: 107-109 Afzelius BA, Eliasson R: Male and female infertility problems in the immotile-cilia syndrome. Eur J Respir Dis 1983; 64(suppl 127): 144-147 Eliasson R, Mossberg B, Camner P, Afzelius BA: The immotilecilia syndrome. N Engl J Med 1977; 297:1-6 Kanematsu T, Matsumata T, Kohno H, Sugimachi K, Inokuchi K: Hepatocellular carcinoma with situ inversus. Cancer 1983; 51: 549-552 Katagener M: Zur pathogenese der Bronchiektasien. Beitr Klin Tuberk 1933;83:489-501 Nielsen MH, Pedersen M, Christensen B, Mygind N: Blind quantitative electron microscopy of cilia from patients with primary ciliary dyskinesia and from normal subjects. Eur J Respir Dis 1983;64(suppl 127): 19-30 Pyscher TJ, Neustein HB: Ciliary dysmorphology. Perspec Pediatr Pathol 1984;8:101-131 Rott HD: Genetics of Kartagener's syndrome. Eur J Respir Dis 1983; 64(suppl 127): 1-4 Rutland J, Cole P: Ciliary dyskinesia. Lancet 1980; 2:859 Sleigh MA, van As A, van den Baan S, et al: Primary ciliary dyskinesia. Lancet 1981; 2:476 Sturgess JM, Chao J, Wong J, Aspin N, Turner JAP: Cilia with defective radial spokes. N Engl J Med 1979; 300:53-56 Sturgess JM, Chao J, Turner JAP: Transposition of ciliary microtubules. N Engl J Med 1980; 303:318-322

Cerebral Toxoplasmosis in an Immunocompromised Host A Precise and Rapid Diagnosis by Electron Microscopy THOMAS T. TANG, M.D., PH.D., JOSEPH M. HARB, PH.D., W. MICHAEL DUNNE, JR., PH.D., ROBERT G. WELLS, MXJ., GLENN A. MEYER, M.D., MICHAEL J. CHUSID, M.D., JAMES T. CASPER, M.D., AND BRUCE M. CAMITTA, M.D.

In immunocompromised patients with cerebral toxoplasmosis, the tachyzpite forms rather than cystic and bradyzoite forms of the protozoon are commonly seen. These tachyzoites are minute, scattered among cellular debris, sometimes lodged inside macrophages and neutrophils, and difficult to visualize by light microscopy, even with special stains. Immunodiagnostic tests may be falsely negative due to inability of the host to produce appropriate antibodies. Isolation of the organism is dangerous because Toxoplasma gondii is highly infective. In this situation, transmission electron microscopy (EM) may be a diagnostic tool of choice. It demonstrates the fine definitive features of the protozoon and can be expedited to give results in five hours. Further

evaluation of EM for diagnosing possible toxoplasmosis in immunocompromised patients is indicated. (Key words: Toxoplasmosis; Toxoplasma gondii; Tachyzoites; Cerebral necrosis; Encephalitis; Immunocompromised hosts; Electron microscopy; Ultrastructure) Am J Clin Pathol 1986; 85: 104-110

Received February 21, 1985; accepted for publication April 19, 1985. Supported in part by the Midwest Atheletes Against Childhood Cancer (MACC Fund). Address reprint requests to Dr. Tang: Milwaukee Children's Hospital, P.O. Box 1997, Milwaukee, Wisconsin 53201.

WITH THE ADVENT of chemotherapy for malignancy, bone marrow and organ transplantations, and acquired immune deficiency syndrome (AIDS), the incidence of opportunistic infection with toxoplasmosis is rising.,-5-6-1215.17.21.23 y ^ conv entional laboratory diagnosis of this

Departments of Pathology, Neurosurgery, Pediatrics, and Radiology, Milwaukee Children's Hospital, Midwest Children's Cancer Center, and Medical College of Wisconsin, Milwaukee, Wisconsin

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Acknowledgments. The authors thank Dr. Ono of Ono Hospital, Oita, and Dr. Y. Shibata of the First Department of Anatomy, Faculty of Medicine, Kyushu University, Fukuoka, Japan, for pertinent discussion, and they thank M. Ohara for editorial assistance.

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Report of a Case A 15-year-old white boy had Philadelphia chromosome-positive chronic myelogenous leukemia. His initial white blood cell (WBC) count was greater than 200,000/^L, and his spleen was massively enlarged. He was treated with busulfan and showed an excellent response. By prognostic formulas his chances for a prolonged chronic phase were small.2,24 Therefore, four months after diagnosis, the patient received a bone marrow transplant from his histocompatible sister. Total body irradiation and high-dose cyclophosphamide were used for transplantation preconditioning. Two months after transplantation, the patient was admitted because of weight loss, jaundice, and low-grade fever. Physical examination revealed moderate hepatosplenomegaly. Pertinent laboratory data included the following: total serum bilirubin, 6.5 mg/dL (indirect, 3.5 mg/dL); SGOT, 320 IU/L; SGPT, 1,280IU/L. The initial impression was hepatitis due to graft-vmw.s-host disease. On the second to fourth hospital days the patient complained of a retroorbital headache. One week after admission a splenectomy was performed because of concern that the enlarged spleen harbored residual leukemic cells. A liver biopsy was obtained at the same time. A cerebrospinalfluid(CSF) specimen at surgery showed 14 WBC (57% lymphocytes; 43% monocytes) and no red blood cells (RBCs) per microliter. The CSF glucose was 65 mg/dL and protein 131 mg/dL. Two days postoperatively the patient became restless and uncoordinated. Vesicular lesions developed on the chest wall and were positive for Tzanck cells. A repeat CSF examination demonstrated 10 WBC (55% lymphocytes; 41% monocytes; 4% polymorphonuclears) per ixL and 149 mg of protein per dL. The possibility of varicella-zoster encephalitis was considered, and intravenous acyclovir was begun. The next day the patient's headache recurred and worsened. He complained of photophobia, appeared to be disoriented, and developed a fever of 39 °C. His left pupil was larger and less reactive than the right. A tonicclonic seizure occurred in the left arm and face, followed by left-sided paresis and numbness. Computed tomography (CT) of the brain with intravenous contrast demonstrated multiple low attenuation lesions in the right cerebral hemisphere with faint rim enhancement and mass

Materials and Methods Microbiology Portions of the biopsied brain tissue were submitted for culture of aerobic and anaerobic bacteria, fungi, mycobacteria, and virus. In addition, a sample of the tissue was forwarded to the State Laboratory of Hygiene for culture of T. gondii in a human embryonic lung cell line. Gram's stains, K.OH preparations, auramine-rhodamine, and trichrome stains were prepared from the specimen and examined for the presence of bacteria, fungi, mycobacteria, and parasites, respectively. Other specimens of the patient submitted for microbiologic evaluation included blood cultures, CSF, spleen and liver biopsies, and swabs of vesicular skin lesions. Immunology Sera obtained from the patient 6 days before and 11 weeks after the bone marrow transplantation were sent to the Parasitic Disease Section at the Center for Disease Control (CDC) for determinations of toxoplasma-specific IgM and IgG titers by indirect immunofluorescence. A CSF specimen obtained 11 weeks after bone marrow transplantation also was submitted for study. Paraffinembedded sections of the brain tissue were forwarded to the CDC and examined for the presence of toxoplasma antigen by direct immunofluorescence. Paraffin embedded sections of brain, liver, and spleen biopsies were submitted to Dr. Conley's laboratory, Palo Alto, California, for detection of T. gondii by the immunoperoxidase method. Light Microscopy The tissues of brain, liver, and spleen biopsies were fixed in 10% neutral buffered formalin and processed according to standard procedures. Sections of tissues, 4 ^m in thickness, were stained with hematoxylin, and eosin (H & E), PAS, Gomori methenamine silver (GMS), Gomori trichrome, Gram's, and Wright-Giemsa stains for examinations for bacteria, fungi, and protozoa.

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protozoan disease in the immunocompromised host may be difficult7"10 because classical toxoplasma cysts with the enclosed bradyzoites may not be formed. Instead, scattered tachyzoites, 2-5 nm in diameter, are encountered more commonly. These fast-developing trophozoites are negative to periodic acid-Schiff (PAS) and Wright-Giemsa stains. Frequently, they are admixed with cellular debris or lodged inside macrophages or neutrophils. Under these circumstances their identification by light microscopy may be extremely difficult.15 Peroxidase-antiperoxidase (PAP) and other immunodiagnostic methods have been useful in demonstrating Toxoplasma gondii.4 However, the antiserum for these methods is available only at a few research centers. Serology and Sabin-Feldman dye tests are timeconsuming and may be falsely negative in patients with immunodeficiency.8,1012 We recently examined biopsied brain tissue from an immunocompromised patient by electron microscopy (EM). Toxoplasma organisms were readily identified. The diagnosis was later confirmed by the PAP method, but serologic tests, immunofluorescent assays, and toxoplasma isolation were noncontributory. The role of EM diagnosis of toxoplasmosis in an immunosuppressed host is discussed.

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effect as evidenced by distortion of the right lateral ventricle (Fig. 1). A 4-cm craniotomy was performed, and biopsies were obtained from the various strata of the occipital lesion with the aid of intraoperative sonography. The specimens were divided for microbiologic cultures and light and electron microscopic examinations. Within two days the diagnosis of T. gondii encephalitis was established by electron microscopy. Therapy with pyrimethamine, sulfadiazine, and folinic acid was begun.6 During the next month after diagnosis the patient's mental status slowly improved. Left hemiparesis and a left homonymous hemianopsia persisted. Repeat CT scans revealed a decrease in the size of the cerebral lesions.

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FIG. 2 (right). Photomicrographs of necrotic brain tissue showing single or multiple round bodies with a halo (arrows) inside polymorphonuclear leukocytes. Trichrome stain (X 1,000).

Electron Microscopy A portion of the brain biopsy was placed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for one hour. The tissue was cut into 1-mm cubes with the aid of a dissecting microscope and rinsed in 0.1 M sodium cacodylate buffer with 5% sucrose, added for osmotic balance. Postfixation was carried out with 1% osmium tetroxide buffered with 0.1 M sodium cacodylate. Tissues were dehydrated in an ascending graded series of methanol, infiltrated with epoxy resin, and embedded in epoxy according to the rapid (stat) schedule previously published.'' One-micron-thick sections were stained with toluidine blue, and pathologically significant areas of the tissue were identified by light microscopy and selected for thin sectioning. Thin sections were collected on rhodiumcopper grids, stained with uranyl acetate and lead citrate, and examined with a JEOL 100-S® electron microscope. Results Microbiology Microscopic examination of stained smears of the brain tissue showed rare neutrophils with intracytoplasmic in-

clusions suggestive of microorganisms. All cultures of the brain tissue were negative after appropriate incubations. Cytomegalovirus was isolated from the spleen biopsy, and varicella-zoster virus was recovered from the skin vesicles. Immunology Analysis of the sera demonstrated no detectable toxoplasma-specific IgM antibody. Toxoplasma-specific IgG titers were elevated in both the pretransplantation (1:4,096) and posttransplantation (1:256) specimens. The posttransplantation CSF contained no detectable toxoplasma-specific IgM and IgG antibodies. Immunofluorescent microscopy of paraffin-embedded sections of the brain tissue were nondiagnostic. Immunoperoxidase stains for T. gondii were positive in brain but negative in liver and spleen tissues. Light Microscopy Sections of brain tissue showed focal necrosis with a spongiform neuropil, small-vessel inflammation, and perivascular hemorrhage. In the necrotic parenchyma there were scattered oval, round, or rarely crescent bodies, 2-5 ^m in diameter, in association with a mixture of neu-

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FlG. 1 (left). Cranial CT scan with intravenous contrast demonstrating a large low attenuation lesion in the right parietooccipital region and a smaller one in the right anterior parietal region. Contrast enhancement is visible at the margins of the lesions. The right lateral ventricle shows a distorted configuration.

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(£U«J FIG. 3. Electron micrograph showing a tachyzoite (arrow) inside the cytoplasm of a neutrophil. The protozoon is ensconced within a parasitophorous vacuole, which gives a halo appearance on light microscopy (XI 7,400).

trophils, microglial cells, macrophages, and lymphocytes. These bodies contained an inconspicuous nucleus and a moderate amount of cytoplasm, which was yellowish brown on H and E, pinkish brown on trichrome, black on GMS, and negative on PAS and Wright-Giemsa stains. Some of these bodies were seen inside macrophages and neutrophils and were often surrounded by a halo (Fig. 2). Many macrophages were laden with lipid droplets. No toxoplasma cysts, viral inclusions, fungi, bacteria, Pneumocystis carinii, amoebae, leukemic cells, or tumors were identified. The diagnosis was probable toxoplasma encephalitis. Sections of the liver showed focal ballooning degeneration of hepatocytes, mild bile stasis, and bile ductular dysplasia suggestive of graft-vmtw-host disease. No leukemic cells or toxoplasma trophozoites were present. Sections of spleen showed diminished malpighian corpuscles, blood sinus congestion, and focal neutrophilia without evidence of leukemia or toxoplasmosis.

Electron Microscopy The brain biopsy contained a moderate number of toxoplasma organisms. An oblique section of a tachyzoite inside a neutrophil is shown in Figures 3 and 4. The tachyzoite measured 2.8 X 1.2 /tm and had a multilayered pellicle with a distinct space between the outer and inner membranes. At the anterior pole and conoid, many microtubules and micronemes were present. The cytoplasm contained abundant nbosomes, a few dense bodies and vacuoles, four to five mottled rhoptries, and rare mitochondria. The entire organism was enclosed inside a parasitophorous vesicle or vacuole, which also contained secretions from the organism. This vacuole corresponds to the halo around each protozoon on light microscopy. A cross-section of a tachyzoite in a lipid-laden macrophage displayed six rhoptries with a distinct lumen and a portion of the nucleus (Fig. 5). It was also ensconced in a parasitophorous vacuole with secretions. Both tachyzoites ap-

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peared undigested and intact and were identified as T. gondii.

other coccidia possess only one or two pairs of balloonlike or club-shaped, solid dense or stippled rhoptries.22 Therefore, electron microscopy (EM) can be specific in identifying T. gondii. Discussion Electron microscopy may be superior to other methods Toxoplasma is a member of Coccidia Order, which also in the diagnosis of toxoplasmosis for many reasons: (1) includes genera of Eimeria, Isospora, Frenkelia, Besnoitia, EM demonstrates the organism directly, whereas imand Sarcocystis.16 Their trophozoites share common ulmunologic methods only indirectly suggest the presence trastructural features of a muhilayered pellicle with miof the organism. (2) Anti-sera for immunofluorescent ascropores and apical complex consisting of polar rings, says are not widely available. Their sensitivity in studying conoid, rhoptries, microtubules, and micronemes.3,22 It immunocompromised hosts with possible toxoplasmosis is mainly on the basis of these fine structures that these is unknown. (3) Serologic studies are often negative in coccidian genera are identified and classified.'8'22 T. gondii immunocompromised patients. Negative immunologic is characterized by the presence of four to eight elongated results should not detract from the value of positive EM sausage- or bat-shaped mottled rhoptries, whereas the findings. (4) With rapid EM processing technics, the di-

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FIG. 4. Electron micrograph showing the oblique section of a tachyzoite with an apical complex (open arrow) consisting of microtubules, micronemes, and aboutfivesausage-like rhoptries (R) which are diagnostic of T. gondii. The cytoplasm is laden with abundant ribosomes, a few dense bodies (d), and vesicles (v) and rare mitochondria. The pellicle (solid arrows) is muhilayered, with a distinct intervening space. Many secretory granules (S) are inside the membrane-lined parasitophorous vacuole, which also contains the tachyzoite. N = segmented nucleus of the neutrophil (X43,000).

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