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Cryptosporidiosis Report of a fatal case complicated by disseminated toxoplasmosis
Cryptosporidiosis Report of a Fatal Case Complicated by Disseminated Toxoplasmosis
GRANT N. STEMMERMANN. TAKUJI HAYASHI, M.D. GARY A. GLOBER, M.D: NOBORU OISHI, M.D. RICHARD I. FRANKEL, M.D.
M.D.
Honolulu. Hawaii
From the Department of Pathology and the Department of Internal Medicine, Kuakini Medical Center, Honolulu, Hawaii. Requests for reprints should be addressed to Dr. Grant N. Stemmermann. Department of Pathology, Kuakini Medical Center, 347 North Kuakini Street, Honolulu, Hawaii 96817. Manuscript accepted October 22, 1979.
A middle-aged woman suffered from chronic diarrhea and malnutrition as a result of a small intestinal infection with a coccidial protozoan-Cryptosporidium. This parasite has been found among a wide range of reptilian, avian and mammalian hosts, but rarely in human beings. This woman ultimately died as a result of disseminated toxoplasmosis of the type usually encountered in an adult whose immune responses were compromised. There was clinical evidence of both humoral and cellular immunologic incompetence, such as depressed levels of complement and immunoglobulins, and decreased T cell reactivity, respectively. The former could be accounted for on the basis of malnutrition causing deficient protein synthesis, and the latter may have been a manifestation of altered function of transformed T cells. The genus Cryptosporidium is a coccidial protozoan of the intestinal tract. It has been identified in a wide range of vertebrates, having been reported in reptiles (snakes) [l], avians [turkeys and chickens) [z] and mammals (rabbits, mice, guinea pigs, dogs, calves, sheep and monkeys) [1,2]. The infection causes enterocolitis in turkeys, guinea pigs and calves, and gastritis in snakes. Coccidia are common intestinal tract parasites composing the suborder Eimeria and are generally transmitted by ingestion of contaminated food or water. The most commonly encountered species of coccidia are Isospora hominis and Isospora belli. Cryptosporidia differ from other coccidia in that they are only found in the microvillus layer of gastrointestinal cells. There are at least six valid species of Cryptosporidia [z]. Species may be identified on the basis of their location within the gut and on the duration of the endogenous life cycle. For example, Cryptosporidium miuris occurs in the stomach, Cryptosporidium tyzzeri in the cecum, Cryptosporidium wrairi in the ileum. C. wrairi, a parasite in guinea pigs, appears to be host-species specific, but whether this specificity is a characteristic of all Cryptosporidia is not known. The life cycle of one of these (C. wrairi) has been clearly described [2]. Trophozoites appear after three to four days of inoculation, followed by two generations of schizonts, the first with eight merozoites and the second with four merozoites. Macro- and microgametes are derived from the second merozoite stage. Presumably microgametes fertilize macrogametes although this has not been observed, nor has an oocyst been identified, Human cryptosporidiosis has been identified in the jejunum of an adult whose immune responses were compromised [3], in the rectum of a 3 year old child [4] and in the small intestine of a nine year old boy with congenital hypogammaglobulinemia [5]. Each patient experi-
October 1980
The Americau
Journal of Medicine
Volume
69
637
CRYPTOSPORIDIOSIS
COMPLICATED
BY DISSEMINATED
TOXOPLASMOSIS-STEMMERMANN
Y I I 9
p 30,000 B I s 3 20,000 cl3 zI
10,000
s
Figure 1. Time trends of total white cell count and lymphocyte count.
enced acute diarrhea. In the adult, this symptom resolved two weeks after cessation of drug-induced irni munosuppression. The child with rectal disease was given supportive therapy only, and the diarrhea ended spontaneously after three weeks. The infection in the boy with hypogammaglobulinemia did not respond to a single course of pyrimethamine and sulfadiazine therapy. Cryptosporidium infection has also been reported in Arabian foals whose responses were immunosuppressed [6]. We wish to document the laboratory findings, the anatomic changes and the lack of response to treatment in another case of cryptosporidial infection of the jejunum and duodenum in a human being. This infection occurred in an immunologically
ultimately
deficient host who
died as a result of severe malnutrition
and
disseminated toxoplasmosis. A brief summary of the clinical aspects of this patient’s illness follows. CASE REPORT A 52 year old woman of .Japaneseancestry first noticed loose bowel movements and a 20 pound weight loss10 months prior to admission, The feces were watery in consistency without signs of steatorrhea or blood and occurred at a frequency of one to four movements a day. Prior to each bowel movement she would experience lower abdominal cramping pain, but the patient denied anorexia, dysphagia, jaundice or fever. As a lifelong Honolulu resident, the patient had made one trip to the mainland (Las Vegas, Nevada] one year prior to the onset of her diarrhea. She worked as a typist and had no exposure to toxins. One brother and a second cousin died of lymphosarcoma. Her only exposure to animals was to a pet dog. She had had the dog for many years and it had suffered no recent illnesses.
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Other than a tonsillectomy as a child she had had no previous hospitalizations. Menopause occurred at the age of 49 years. The patient was admitted to the hospital on April 28.1978. She was thin, but alert and in no acute distress. She was normotensive without orthostatis hypotension. There was no peripheral lymphadenopathy. Her abdomen was tympanitic. There was no hepatosplenomegaly. Initial diagnostic studies indicated that she had lymphocytosis (81 percent of 9,900/mm3 white blood cells] and that 40 percent of the lymphocytes were atypical. Subsequent counts showed persistent lymphocytosis with a terminal increase in the number of white blood cells and lymphocytes (Figure I). On admission, the hematocrit value was 38 percent with a mean corpuscular hemoglobin concentration of 35 percent and a mean corpuscular volume of 107 p. Serum glutamic oxaloacetic transaminase @GOT) was 90 U, serum glutamic pyruvic transaminase (SGPT) was 50 U and creatinine phosphokinase was normal. Alkaline phosphatase was 30 U (normal, 9 to 35 U), total bilirubin 0.6 mg/lOO ml and a glutamyl transpeptidase was 20 U (normal less than 40 U). Uric acid was 4.3 mg/lOO ml, albumin 4.2 g/100 ml and globulin 3.1 g/100 ml. Serum vitamin BQ was 250 pg/ml (normal 200 to 900 pg/ml) and folate 4.1 mg/ml [normal 4 to 20 ms/ml]. Triiodothyronine resin uptake was 41.5 percent (normal 35 to 45 percent] and thyroxine by radioimmunoassay was 8.2 pg/lOO ml [normal 4.5 to 11.5 r(g/lOO ml). A 24-hour urine 5-hydroxyindole acetic acid determination was within normal limits. No white blood cells were seen in the stool, and an initial urinalysis did not disclose any abnormalities. Serum cholesterol was 100 mg/dl (normal 160 to 250 mg/dl), triglyceride 59 mg/dl (normal 10 to 160 mg/dl) and magnesium was low at 1.2 mg/dl (normal 1.3 to 2.6 mg/dl). Serum carotene was 8 pg/dl (normal 50 to 250 pg/dl] and prothrombin time was 60 percent. Stool examinations were negative for parasites on numerous occasions as were examinations of duodenal aspirates. A vasoactive intestinal polypeptide level was 150 pg/ml [normal 3 to 36 pg/ml). A roentgenographic series of the small bowel showed that the proximal jejunum had an increased caliber and coarse plicae. Barium enema and protoscopy revealed no abnormalities. Computerized tomography failed to reveal enlargement of retroperitoneal lymph nodes, and a lymphangiogram was within normal limits. A technitium sulfur colloid liver scan revealed a liver measuring 13 cm in the mid-clavicular line. Colloidal distribution was not uniform, but there were no discrete focal lesions. The spleen measured 9 cm and the spleen to liver ratio was normal at 0.69. Hepatitis B surface antigen was not present and the liver biopsy revealed only fatty metamorphosis. A jejunal biopsy specimen showed villous atrophy. Many small parasites identified as Cryptosporidium were found on the luminal surface of the villous epithelium. These organisms were not recovered from the feces or the duodenal aspirate. Antibodies against Toxoplasma gondii were estimated to be present at a titer of 1:512 by the indirect hemagglutination technique on May 3.1978, and at a level of 1:256 on July 11. The antibody titers against cytomegalic virus were 1:64 on two occasions and against Epstein-Barr virus were 1:lOO and 1:200, as estimated by the indirect fluorescent antibody technique. Evidence of an impaired immunologic status is summarized by the following abnormal findings:
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TABLE
I
Date April 1978 June 1978 July 1978 August 1978 l
Results of Lymphocyte
COMPLICATED
Stimulation
BY DISSEMINATED
TOXOPLASMOSIS-STEMMERMANN
ET AI..
Test*
ConcanavallnA Phylohemag@tinln PokeweedMilogen 19 19 4
3
18 244 4 4
21 14
1 1
Number of mitogen events per 2.5 X lo5 cells.
Immunoglobulin G was less than 800 mg/lOO ml in four of five analyses (range 600 to 950 mg/lOO ml); immunoglobulin M was more than 70 mg/lOO ml in one of five analyses [range 63 to 98 mg/lOO ml]; immunoglobulin A, in each of five analyses, was within normal limits (range 131 to 192 mg/lOO ml). The results of the lymphocyte stimulation tests are shown in Table I. The normal range for concanavalin A is 38 to 1% mitogen responses among 250,000 cells, for phytohemagglutinin 61 to 173 and for pokeweed mitogen 7 to 106. The normal range for B cells is 27 to 49 percent. In our patient it was 9.7 to 25 percent. The normal range for T cells is 36 to 60 percent. In our patient, the range was 36 to 60 percent. In May, there was no response to Candida, dermatophytin, mumps or purified protein derivative skin tests. In July, there was no response to mumps, varidase and purified protein derivative skin tests. There was an erythematous reaction (l+) to 2 mg DNCB sensitization; no reaction to 0.025 to 0.1 mg. In April 1978, the third component of complement (C3c) was 63 mg/lOO ml and in May it was 65 mg/lOO ml (normal range. 62 to 212 mg/lOO ml). In April 1978, the fourth component of complement (C4) was 40 mg/lOO ml and in May it was 24 mg/lOO ml (normal range 20 to 50 mg/lOO ml). In June 1978, total complement was 32 U/ml (CH 50; normal range 48 to 103 U/ml]. C3 activator was 5.8 mg/lOO ml [normal range 12 to 30 mg/lOO ml]. The cryptosporidial infection resisted a wide variety of antibiotic agents. The antibiotic therapy may be summarized as follows: (1) Metronidazole, 250 mg, three times a day from April 28 to May 10. There was no response to this regimen. (2) Sulfamethoxazole, 800 mg, and trimethoprim, 160 mg, every 6 houm from May 10 to May 22. There was no clinical response although a serum sulfamethoxazole level on the third day of treatment was at normal therapeutic levels 110.2 mg/dl). (3) Ryrimethamine in an initial dose of 50 mg on May 30, followed by 25 mg daily from June 1 to June 15. A serum level on June 7 was 0.4 &ml-within the normal therapeutic range for this drug. (4) Sulfadiazine, 750 mg every 6 hours from May 31 to June 15. The sulfadiazine level was 8.7 mg/dl on June 7 and 9 mg/dl on June 10, again demonstrating adequate absorption and therapeutic levels. (5) Brief courses of levamisol from June 30 to July 3 and from July 13 to July 15 yielded no clinical response. (61 Amphotericin B was given intravenously from July 31 to August 23 for a total dose of 466 mg. Concomittant oral’ amphotericin B was given every 8 hours from August 3 to August 23 for a total dose of 549 mg. The serum amphotericin B level was measured on two occasions with levels of 0.32 ug/ml and 0.16 ug/ml. There was no response to this therapy. Repeat jejunal and duodenal biopsies indicated that the cryptosporidial organisms increased in number throughout the
October
Figure 2. The jejunum showing total villus atrophy, elongation of the crypts and increased mitoses. The lamina propria is infiltrated with lymphocytes, a few plasma cells and histiocytes. Hematoxylin and eosin stain; magnification X 80, reduced by 17 percent. Insert, surface epithelial layer with Cryptosporidia. Hematoxylin and eosin stain; magnification X 600, reduced by 77 percent.
period of antibiotic therapy. Initially the patient was given an elemental diet, but her diarrhea did not lessen. She did not respond to the administration of cholestyramine or to a trial of an oral mixture of glucose and actively transported amino acids. Her bowel movements became so watery and explosive that eventually she could only be supported by parenteral hyperalimentation. She died in August 1978,22 months after the onset of her loose bowel movements. Terminally, generalized lymphadenopathy, renal failure and pulmonary insufficiency developed. She weighed 88 pounds on admission and one day before her death she weighed 49 pounds. The serum creatinine level had risen from 0.7 mg/lOO ml on admission to 4.4 mg/lOO ml. Albumin decreased from 3.9 to 2.6, the alkaline phosphatase level had risen from 30 to 120 U, SCOT from 81 to 110 U. and the prothrombin time had de-
Figure 3. Low-power electron micrograph showing Cryptosporidia at various stages of development. T = trophozoite, S = schizont, M = macrogamete. Magnification X 4,700.
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Figure 4. Trophozoite with a single nucleus with a large nucleolus (N), numerous free ribosomes, a few conoids (C). Note the absence of mitochondria and poorly developed attachment zone. Cryptosporidium is covered with a parasitophorous membrane (P); attachment to an enterocyte is marked by a dense band. Magnification X 28,000.
creased from 30 percent to 33 percent. The platelet count was 56,000/mm3, but there was no laboratory evidence for disseminated intravascular coagulation. Cholesterol was stable at 119 mg/lOO ml.
ET AL,
zoites were covered with a parasitophorous membrane (Figure 4) and had a large nucleus, many free ribosomes and conoid structures, but no mitochondria or differentiated comb-like attachment zones. More mature trophozoites had well-developed granular endoplasmic reticulum, vacuoles and occasional dense bodies. In the schizont stage (Figure 5) trophozoites formed a well-developed parasitophorous space and had multiple anucleolar nuclei, a well-developed granular endoplasmic reticulum and occasional vacuoles. Merozoites had small anucleolar nuclei with cytoplasmic vacuoles and dense bodies. They were surrounded by a pellicle. Some merozoites were in the stage of release from the parasitophorous membranes. Macrogametes had attachment zones, anucleolar nuclei, a few dense bodies and “polysaccharide” granules arranged at the periphery of the cytoplasm. The relation of the parasitophorous membrane to the enterocytic microvillus was not clear-cut. The outer aspect of the membrane was covered with a small amount of glycocaly+. There was an indistinct, discontinuous, membranous or filamentous structure between the two unit membranes of the parasitophorous membrane, especially at the basal and lateral aspects. It disappeared as it was traced toward the luminal side. At the site of attachment, a dense band separated the Cryptosporidium and enterocytes that was continuous with the outer parasitophorous membrane. Above this band the Cryptosporodium was equipped with comb-like folded membranes (Figure 6).
AUTOPSY FINDINGS The axillary, inguinal, periaortic, mesenteric and mediastinal lymph nodes were all enlarged, but none exceeded 2.0 cm in diameter. On histologic examination nodes from all sites showed either focal or total acute necrosis. The areas of necrosis contained clumps of fibrin and nuclear fragments. The viable portions of the nodes had a varied cell population with
SURGICAL PATHOLOGY The jejunal villi were short and blunt, the crypts longer and wider than normal (Figure 31. The lamina propria contained many lymphocytes and monocytes. Many small parasites (approximately z to 3 p in diameter) were attached to luminal surfaces of the epithelial cells at the tips of the villi. The jejunum and duodenum were biopsied on several occasions in order to assess the efficacy of various drugs. These examinations indicated that the parasites increased in number with time. There was a parallel increase in the extent ofvillus atrophy. This was seen in its most extreme form in the duodenum nine days prior to death. Gastric biopsy specimens taken on this occasion indicated that cryptosporidial infection ended abruptly at the gastroduodenal junction. Electron microscopy revealed numerous Cryptosporidia at various stages of development. They were similar to those described in previous reports [5,‘i’]. Maturing trophozoites, schizonts, merozoites and early macrogametes were identified (Figure 3). They were embedded in microvilli and attached to enterocytes, thereby pushing microvilli away. The enterocytes were indented and had widened terminal webs that were devoid of rootlets of microvilli. Each Cryptosporidium was covered by a parasitophorous membrane. Maturing tropho-
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Flgure 5. Schizont (S) and’macrogamete (M). Schizont is surrounded by a parasitophorous membrane with a widened space and has three anucleolar nuclei. Macrogamete has a welldeveloped attachment zone, and granular endoplasmic reticulum, an anucleolar nucleus, a few dense bodies, and peripherally arranged “polysaccharide” granules. Magnification X 13,000.
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BY DISSEMINATED
ET AL.
dense aggregates of lymphocytes and monocytes. This change was associated with toxoplasma1 organisms and was most intense at the corticomedullary junctions; in this region, it was associated with necrosis of the tubular epithelium of Henle’s loops. The gastric mucosa was dark red and covered by thick plaques of friable brown material. There were extensive areas of intramural necrosis similar to those in the lungs and lymph nodes. The areas of necrosis involved both submucosa and the external muscle coat, and contained many Toxoplasma organisms. Similar zones of necrosis were also encountered in the small and large bowels. The intestinal epithelium was entirely lost as a result of autolysis; hence no cryptosporidial organisms could be identified at autopsy. The right parietal lobe of the cerebrum contained a 1 cm zone of grossly apparent necrosis. All portions of the central nervous system and the posterior pituitary contained many Toxoplasma organisms. There were many microinfarcts containing lumps of Toxoplasma organisms measuring an average of 11 pm. Electron microscopy showed a disrupted cyst wall and many Toxoplasma. These parasites had double plasma membranes, toxonemes, osmiophilic vacuoles, mitochondria and conoids. Other organs, including the bone marrow, thymus, thyroid and parathyroid glands, showed no pathologic changes. The pancreas, adrenal glands and uterus were without major structural alterations except for perivascular collections of lymphocytes and monocytes. In summary, the immediate cause of this patient’s death was an overwhelming dissemination of Toxoplasma manifested by necrotizing encephalitis, lymphadenitis, pneumonitis and intramural gastroenteritis. The underlying cause of death was severe malnutrition due to cryptosporidial infection of the small intestine.
Figure 6. Attachment zone below the comb-like folded membranes showing a dense band and thin band of the parasitophorous membrane separating Cryptosporidium and enterocyte. The dense band is continuous with the outer layer of the parasitophorous membrane. Note the absence of microvtllous rootlets below the attachment zone. Magnification X 58,000. small and large lymphocytes, histiocytes, plasma cells and neutrophils. There was no suggestion of neoplastic change in any node. Groups of small organisms resembling T. gondii were present in the stromal portions of intact nodes and in the subcapsular lymphatic channels. The lungs contained firm, nonaerated oval gray areas surrounded by a distinct zone of hyperemia. These were most numerous in the lower lobes and displayed the same type of necrosis as that found in the lymph nodes. The necrosis centered about the walls of small pulmonary arteries. The adjacent alveolar spaces were filled with fibrin and nuclear fragments. The histiocytes of the lung contained groups of organisms which ultrastructural analysis identified as Toxoplasma. They had double plasma membranes, mitochondria, toxonemes, conoids and osmiophilic vacuoles (Figure 7). This appearance is consistent with previous reports of this organism [8]. Positive immunofluorescent labeling with Toxoplasma antibody was achieved for these pulmonary parasites, according to the method of Huang et al. [9]. The heart was not unusual to the naked eye and weighed 300 g. The muscle fibers of the left ventricle contained aggregates of toxoplasma1 parasites associated with interstitial aggregates of lymphocytes and monocytes. The liver was greatly enlarged (2,075 g) and showed extensive fatty change but no areas of inflammation or necrosis. The spleen weighed 200 g and the pulp contained many histiocytes showing erythrophagocytosis. The kidneys appeared swollen and weighed 200 and 225 g. Their capsular surfaces were smooth but mottled. The interstitial tissue was infiltrated with
October
TOXOPLASMOSIS-STEMMERMANN
COMMENT!3 recorded cases of human small intestinal cryptosporidiosis occurred in immunosuppressed hosts.
The previously
Figure 7. Higher-power electron micrograph showing double plasma membranes, mitochondria (M), polar conoids (C) and toxonemes (T). Magnification X 34.000.
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In one patient the infection ended spontaneously when immunosuppression was corrected [3], but an infection in a child with hypogammaglobulinemia did not respond to medication [4]. Our patient had received no medication known to induce immunosuppression, nor could any illness be identified to account for an immunosuppressed state (e.g., Hodgkin’s disease, Sjiigren’s disease, lymphosarcoma). The patient had a brother and a second cousin who died with lymphosarcoma. Although she had a normal white blood cell count, she displayed a conspicuous lymphocytosis (60 to 81 percent] throughout the last six months of her life, and 25 to 50 percent of her lymphocytes were atypical. Numerous bone marrow examinations were performed, yet none gave any evidence of lymphosarcoma or leukemia. The autopsy also failed to establish this diagnosis. The sudden increase in the number of lymphocytes in the last three weeks of life might have been a response to the disseminated toxoplasmosis or an early manifestation of leukemia. There was evidence of both humoral and. cellular immunologic incompetence-depressed levels of complement and immunoglobulins and decreased T cell reactivity, respectively. The former could be accounted for on the basis of malnutrition causing deficient protein synthesis, the latter on the basis.of altered function of transformed T cells. The antibody levels against cytomegalic virus and Epstein-Barr virus indicate that the patient had previously been infected by these viruses. Cytomegalic virus infection is frequently encountered in the immunologically compromised host, yet there was no evidence of active infection by this virus at autopsy. The decrease in the titer of T. gondii antibody probably reflects a decrease in the production of humoral antibody due to malnutrition. Disseminated toxoplasmosis of the type found in this patient is not usually encountered among adults except when they are under treatment with agents causing immunosuppression [lO,ll]. The synchronous occurrence of cryptosporidiosis and dissem-
ET AL
inated toxoplasmosis suggests that these diseases are subject to similar immunologic restraints. The cryptosporidiosis of immunodeficient Arabian foals [6] would appear to be a good model of the disease in human subjects. The immunodeficiency of these animals is an autosomal recessive disease characterized by lack of development of B and T lymphocytes [x]. It resembles the combined immunodeficiency of the Swiss type that is characterized by lymphopenia, rather than lymphocytosis as in our case. Therapeutic immunosuppression also results in combined immunodeficiency. Neither equine cryptosporidiosis nor any of the cases in human subjects, therefore, offers a clue as to the specific immunologic defect favoring infection by this organism. The disseminated necrotizing inflammation discovered at necropsy posed a problem of interpretation at the light microscopic level. Foci of necrosis in t$e lungs, gut wall, lymph nodes and brain contained organisms resembling T. gondii. We were not entirely certain whether an oocystic stage of Cryptosporidium could be ruled out, even though neither cryptosporidial oocysts nor disseminated cryptosporidiosis have been described. Ultrastructural and immunofluorescent studies of the organisms in the lungs and central nervous system, however, clearly identify them as toxoplasma1 in type. They contained mitochondria and polar conoids, neither of which is found in Cryptosporidia [7]. ACKNOWLEDGMENT
We wish to express our gratitude to Dr. W. M. Meyers, Chief, Microbiology Division, Armed Forces Institute of Pathology, for his assistance in the identification of the cryptosporidial organism: to Dr. 1. K. Frenkel, Department of Pathology, University of Kansas Medical Center, for his assistance in the identification of the toxoplasma1 organism, and to Dr. D. A. Stevens, University of Stanford, School of Medicine, Palo Alto, California and Dr. G. S. Visvesvara, C.D.C., Atlanta, Georgia, for their advice and assistance.
REFERENCES 1. Brownstein DG. Strandberg JD. Montoli RJ, Bush M, Former J: Cryptosporidium in snakes with hypertrophic gastritis. Vet Path01 1977; 14: 606. 2. Vetterling JM, Jervis HR. Merrill TG, Sprinz H: Cryptosporidium wrairi from the guinea pig Cavia porcellus with an emendation of the genus. J Protozool1971; 18: 243. 3. Meisel JL, Perera DR; Meligro C, Rubin CE: Overwhelming watery diarrhea associated with a cryptosporidium in an immunosuppressed patient. Gastroenterology 1976; 70: 1156. 4. Nime FA, Burek JD, Page DL, Holscher MA, Yardley JH: Acute enterocolitis in a human being infected with the protozoan crfrptosporidium. Gastroenterology 1976; 79: 592. 5. Lasser KH, Lewin KJ, Ryning Fw: Cryptosporidial enteritis in a patient with congenital hypogammaglobulinemia. Hum Path01 1979; 10: 234. 6. Snyder SP, England JJ, McChesney AE: Cryptosporidiosis
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&i immunodeficient arabian foals. Vet Patbol 1978; 15: 12. 7. Vetterling JM, Takeuchi A, Madden PA: Ultrastructure of Cryptosporidium wrairi from the guinea pig. J Protozool 1971;18: 248. 8. Callaway CS, Walls KW, Hicklin MD: Electron microscopic studies of toxoplasma gondii. Arch Path01 1968; 86: 484. 9. Huan SN, Minassian H, More JD: Application of immuno ff uorescent staining on paraffin sections improve&by try sin digestion. Lab Invest 1976; 35: 383. 10. Fren t: el JK, Nelson BM, Arias-Stella J: Immunosuppression and toxoplasmic encephalitis. Hum Path01 1975: 6: 97. 11. Frenkel JK, Amore M. Larsen W: Immune competence in a patient with Hodgkin’s disease and relapsing toxoplasmosis. Infection 1978; 6: 3. 12. McGuire TC, Banks KL, Davis WC: Alterations of the thymus and other lymphoid tissue in young horses with combined immunodeficiency. Am J Path01 197fi; 84: 39.
Volume
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GRANT N. STEMMERMANN. TAKUJI HAYASHI, M.D. GARY A. GLOBER, M.D: NOBORU OISHI, M.D. RICHARD I. FRANKEL, M.D.
M.D.
Honolulu. Hawaii
From the Department of Pathology and the Department of Internal Medicine, Kuakini Medical Center, Honolulu, Hawaii. Requests for reprints should be addressed to Dr. Grant N. Stemmermann. Department of Pathology, Kuakini Medical Center, 347 North Kuakini Street, Honolulu, Hawaii 96817. Manuscript accepted October 22, 1979.
A middle-aged woman suffered from chronic diarrhea and malnutrition as a result of a small intestinal infection with a coccidial protozoan-Cryptosporidium. This parasite has been found among a wide range of reptilian, avian and mammalian hosts, but rarely in human beings. This woman ultimately died as a result of disseminated toxoplasmosis of the type usually encountered in an adult whose immune responses were compromised. There was clinical evidence of both humoral and cellular immunologic incompetence, such as depressed levels of complement and immunoglobulins, and decreased T cell reactivity, respectively. The former could be accounted for on the basis of malnutrition causing deficient protein synthesis, and the latter may have been a manifestation of altered function of transformed T cells. The genus Cryptosporidium is a coccidial protozoan of the intestinal tract. It has been identified in a wide range of vertebrates, having been reported in reptiles (snakes) [l], avians [turkeys and chickens) [z] and mammals (rabbits, mice, guinea pigs, dogs, calves, sheep and monkeys) [1,2]. The infection causes enterocolitis in turkeys, guinea pigs and calves, and gastritis in snakes. Coccidia are common intestinal tract parasites composing the suborder Eimeria and are generally transmitted by ingestion of contaminated food or water. The most commonly encountered species of coccidia are Isospora hominis and Isospora belli. Cryptosporidia differ from other coccidia in that they are only found in the microvillus layer of gastrointestinal cells. There are at least six valid species of Cryptosporidia [z]. Species may be identified on the basis of their location within the gut and on the duration of the endogenous life cycle. For example, Cryptosporidium miuris occurs in the stomach, Cryptosporidium tyzzeri in the cecum, Cryptosporidium wrairi in the ileum. C. wrairi, a parasite in guinea pigs, appears to be host-species specific, but whether this specificity is a characteristic of all Cryptosporidia is not known. The life cycle of one of these (C. wrairi) has been clearly described [2]. Trophozoites appear after three to four days of inoculation, followed by two generations of schizonts, the first with eight merozoites and the second with four merozoites. Macro- and microgametes are derived from the second merozoite stage. Presumably microgametes fertilize macrogametes although this has not been observed, nor has an oocyst been identified, Human cryptosporidiosis has been identified in the jejunum of an adult whose immune responses were compromised [3], in the rectum of a 3 year old child [4] and in the small intestine of a nine year old boy with congenital hypogammaglobulinemia [5]. Each patient experi-
October 1980
The Americau
Journal of Medicine
Volume
69
637
CRYPTOSPORIDIOSIS
COMPLICATED
BY DISSEMINATED
TOXOPLASMOSIS-STEMMERMANN
Y I I 9
p 30,000 B I s 3 20,000 cl3 zI
10,000
s
Figure 1. Time trends of total white cell count and lymphocyte count.
enced acute diarrhea. In the adult, this symptom resolved two weeks after cessation of drug-induced irni munosuppression. The child with rectal disease was given supportive therapy only, and the diarrhea ended spontaneously after three weeks. The infection in the boy with hypogammaglobulinemia did not respond to a single course of pyrimethamine and sulfadiazine therapy. Cryptosporidium infection has also been reported in Arabian foals whose responses were immunosuppressed [6]. We wish to document the laboratory findings, the anatomic changes and the lack of response to treatment in another case of cryptosporidial infection of the jejunum and duodenum in a human being. This infection occurred in an immunologically
ultimately
deficient host who
died as a result of severe malnutrition
and
disseminated toxoplasmosis. A brief summary of the clinical aspects of this patient’s illness follows. CASE REPORT A 52 year old woman of .Japaneseancestry first noticed loose bowel movements and a 20 pound weight loss10 months prior to admission, The feces were watery in consistency without signs of steatorrhea or blood and occurred at a frequency of one to four movements a day. Prior to each bowel movement she would experience lower abdominal cramping pain, but the patient denied anorexia, dysphagia, jaundice or fever. As a lifelong Honolulu resident, the patient had made one trip to the mainland (Las Vegas, Nevada] one year prior to the onset of her diarrhea. She worked as a typist and had no exposure to toxins. One brother and a second cousin died of lymphosarcoma. Her only exposure to animals was to a pet dog. She had had the dog for many years and it had suffered no recent illnesses.
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ET AL.
Other than a tonsillectomy as a child she had had no previous hospitalizations. Menopause occurred at the age of 49 years. The patient was admitted to the hospital on April 28.1978. She was thin, but alert and in no acute distress. She was normotensive without orthostatis hypotension. There was no peripheral lymphadenopathy. Her abdomen was tympanitic. There was no hepatosplenomegaly. Initial diagnostic studies indicated that she had lymphocytosis (81 percent of 9,900/mm3 white blood cells] and that 40 percent of the lymphocytes were atypical. Subsequent counts showed persistent lymphocytosis with a terminal increase in the number of white blood cells and lymphocytes (Figure I). On admission, the hematocrit value was 38 percent with a mean corpuscular hemoglobin concentration of 35 percent and a mean corpuscular volume of 107 p. Serum glutamic oxaloacetic transaminase @GOT) was 90 U, serum glutamic pyruvic transaminase (SGPT) was 50 U and creatinine phosphokinase was normal. Alkaline phosphatase was 30 U (normal, 9 to 35 U), total bilirubin 0.6 mg/lOO ml and a glutamyl transpeptidase was 20 U (normal less than 40 U). Uric acid was 4.3 mg/lOO ml, albumin 4.2 g/100 ml and globulin 3.1 g/100 ml. Serum vitamin BQ was 250 pg/ml (normal 200 to 900 pg/ml) and folate 4.1 mg/ml [normal 4 to 20 ms/ml]. Triiodothyronine resin uptake was 41.5 percent (normal 35 to 45 percent] and thyroxine by radioimmunoassay was 8.2 pg/lOO ml [normal 4.5 to 11.5 r(g/lOO ml). A 24-hour urine 5-hydroxyindole acetic acid determination was within normal limits. No white blood cells were seen in the stool, and an initial urinalysis did not disclose any abnormalities. Serum cholesterol was 100 mg/dl (normal 160 to 250 mg/dl), triglyceride 59 mg/dl (normal 10 to 160 mg/dl) and magnesium was low at 1.2 mg/dl (normal 1.3 to 2.6 mg/dl). Serum carotene was 8 pg/dl (normal 50 to 250 pg/dl] and prothrombin time was 60 percent. Stool examinations were negative for parasites on numerous occasions as were examinations of duodenal aspirates. A vasoactive intestinal polypeptide level was 150 pg/ml [normal 3 to 36 pg/ml). A roentgenographic series of the small bowel showed that the proximal jejunum had an increased caliber and coarse plicae. Barium enema and protoscopy revealed no abnormalities. Computerized tomography failed to reveal enlargement of retroperitoneal lymph nodes, and a lymphangiogram was within normal limits. A technitium sulfur colloid liver scan revealed a liver measuring 13 cm in the mid-clavicular line. Colloidal distribution was not uniform, but there were no discrete focal lesions. The spleen measured 9 cm and the spleen to liver ratio was normal at 0.69. Hepatitis B surface antigen was not present and the liver biopsy revealed only fatty metamorphosis. A jejunal biopsy specimen showed villous atrophy. Many small parasites identified as Cryptosporidium were found on the luminal surface of the villous epithelium. These organisms were not recovered from the feces or the duodenal aspirate. Antibodies against Toxoplasma gondii were estimated to be present at a titer of 1:512 by the indirect hemagglutination technique on May 3.1978, and at a level of 1:256 on July 11. The antibody titers against cytomegalic virus were 1:64 on two occasions and against Epstein-Barr virus were 1:lOO and 1:200, as estimated by the indirect fluorescent antibody technique. Evidence of an impaired immunologic status is summarized by the following abnormal findings:
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TABLE
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Date April 1978 June 1978 July 1978 August 1978 l
Results of Lymphocyte
COMPLICATED
Stimulation
BY DISSEMINATED
TOXOPLASMOSIS-STEMMERMANN
ET AI..
Test*
ConcanavallnA Phylohemag@tinln PokeweedMilogen 19 19 4
3
18 244 4 4
21 14
1 1
Number of mitogen events per 2.5 X lo5 cells.
Immunoglobulin G was less than 800 mg/lOO ml in four of five analyses (range 600 to 950 mg/lOO ml); immunoglobulin M was more than 70 mg/lOO ml in one of five analyses [range 63 to 98 mg/lOO ml]; immunoglobulin A, in each of five analyses, was within normal limits (range 131 to 192 mg/lOO ml). The results of the lymphocyte stimulation tests are shown in Table I. The normal range for concanavalin A is 38 to 1% mitogen responses among 250,000 cells, for phytohemagglutinin 61 to 173 and for pokeweed mitogen 7 to 106. The normal range for B cells is 27 to 49 percent. In our patient it was 9.7 to 25 percent. The normal range for T cells is 36 to 60 percent. In our patient, the range was 36 to 60 percent. In May, there was no response to Candida, dermatophytin, mumps or purified protein derivative skin tests. In July, there was no response to mumps, varidase and purified protein derivative skin tests. There was an erythematous reaction (l+) to 2 mg DNCB sensitization; no reaction to 0.025 to 0.1 mg. In April 1978, the third component of complement (C3c) was 63 mg/lOO ml and in May it was 65 mg/lOO ml (normal range. 62 to 212 mg/lOO ml). In April 1978, the fourth component of complement (C4) was 40 mg/lOO ml and in May it was 24 mg/lOO ml (normal range 20 to 50 mg/lOO ml). In June 1978, total complement was 32 U/ml (CH 50; normal range 48 to 103 U/ml]. C3 activator was 5.8 mg/lOO ml [normal range 12 to 30 mg/lOO ml]. The cryptosporidial infection resisted a wide variety of antibiotic agents. The antibiotic therapy may be summarized as follows: (1) Metronidazole, 250 mg, three times a day from April 28 to May 10. There was no response to this regimen. (2) Sulfamethoxazole, 800 mg, and trimethoprim, 160 mg, every 6 houm from May 10 to May 22. There was no clinical response although a serum sulfamethoxazole level on the third day of treatment was at normal therapeutic levels 110.2 mg/dl). (3) Ryrimethamine in an initial dose of 50 mg on May 30, followed by 25 mg daily from June 1 to June 15. A serum level on June 7 was 0.4 &ml-within the normal therapeutic range for this drug. (4) Sulfadiazine, 750 mg every 6 hours from May 31 to June 15. The sulfadiazine level was 8.7 mg/dl on June 7 and 9 mg/dl on June 10, again demonstrating adequate absorption and therapeutic levels. (5) Brief courses of levamisol from June 30 to July 3 and from July 13 to July 15 yielded no clinical response. (61 Amphotericin B was given intravenously from July 31 to August 23 for a total dose of 466 mg. Concomittant oral’ amphotericin B was given every 8 hours from August 3 to August 23 for a total dose of 549 mg. The serum amphotericin B level was measured on two occasions with levels of 0.32 ug/ml and 0.16 ug/ml. There was no response to this therapy. Repeat jejunal and duodenal biopsies indicated that the cryptosporidial organisms increased in number throughout the
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Figure 2. The jejunum showing total villus atrophy, elongation of the crypts and increased mitoses. The lamina propria is infiltrated with lymphocytes, a few plasma cells and histiocytes. Hematoxylin and eosin stain; magnification X 80, reduced by 17 percent. Insert, surface epithelial layer with Cryptosporidia. Hematoxylin and eosin stain; magnification X 600, reduced by 77 percent.
period of antibiotic therapy. Initially the patient was given an elemental diet, but her diarrhea did not lessen. She did not respond to the administration of cholestyramine or to a trial of an oral mixture of glucose and actively transported amino acids. Her bowel movements became so watery and explosive that eventually she could only be supported by parenteral hyperalimentation. She died in August 1978,22 months after the onset of her loose bowel movements. Terminally, generalized lymphadenopathy, renal failure and pulmonary insufficiency developed. She weighed 88 pounds on admission and one day before her death she weighed 49 pounds. The serum creatinine level had risen from 0.7 mg/lOO ml on admission to 4.4 mg/lOO ml. Albumin decreased from 3.9 to 2.6, the alkaline phosphatase level had risen from 30 to 120 U, SCOT from 81 to 110 U. and the prothrombin time had de-
Figure 3. Low-power electron micrograph showing Cryptosporidia at various stages of development. T = trophozoite, S = schizont, M = macrogamete. Magnification X 4,700.
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Figure 4. Trophozoite with a single nucleus with a large nucleolus (N), numerous free ribosomes, a few conoids (C). Note the absence of mitochondria and poorly developed attachment zone. Cryptosporidium is covered with a parasitophorous membrane (P); attachment to an enterocyte is marked by a dense band. Magnification X 28,000.
creased from 30 percent to 33 percent. The platelet count was 56,000/mm3, but there was no laboratory evidence for disseminated intravascular coagulation. Cholesterol was stable at 119 mg/lOO ml.
ET AL,
zoites were covered with a parasitophorous membrane (Figure 4) and had a large nucleus, many free ribosomes and conoid structures, but no mitochondria or differentiated comb-like attachment zones. More mature trophozoites had well-developed granular endoplasmic reticulum, vacuoles and occasional dense bodies. In the schizont stage (Figure 5) trophozoites formed a well-developed parasitophorous space and had multiple anucleolar nuclei, a well-developed granular endoplasmic reticulum and occasional vacuoles. Merozoites had small anucleolar nuclei with cytoplasmic vacuoles and dense bodies. They were surrounded by a pellicle. Some merozoites were in the stage of release from the parasitophorous membranes. Macrogametes had attachment zones, anucleolar nuclei, a few dense bodies and “polysaccharide” granules arranged at the periphery of the cytoplasm. The relation of the parasitophorous membrane to the enterocytic microvillus was not clear-cut. The outer aspect of the membrane was covered with a small amount of glycocaly+. There was an indistinct, discontinuous, membranous or filamentous structure between the two unit membranes of the parasitophorous membrane, especially at the basal and lateral aspects. It disappeared as it was traced toward the luminal side. At the site of attachment, a dense band separated the Cryptosporidium and enterocytes that was continuous with the outer parasitophorous membrane. Above this band the Cryptosporodium was equipped with comb-like folded membranes (Figure 6).
AUTOPSY FINDINGS The axillary, inguinal, periaortic, mesenteric and mediastinal lymph nodes were all enlarged, but none exceeded 2.0 cm in diameter. On histologic examination nodes from all sites showed either focal or total acute necrosis. The areas of necrosis contained clumps of fibrin and nuclear fragments. The viable portions of the nodes had a varied cell population with
SURGICAL PATHOLOGY The jejunal villi were short and blunt, the crypts longer and wider than normal (Figure 31. The lamina propria contained many lymphocytes and monocytes. Many small parasites (approximately z to 3 p in diameter) were attached to luminal surfaces of the epithelial cells at the tips of the villi. The jejunum and duodenum were biopsied on several occasions in order to assess the efficacy of various drugs. These examinations indicated that the parasites increased in number with time. There was a parallel increase in the extent ofvillus atrophy. This was seen in its most extreme form in the duodenum nine days prior to death. Gastric biopsy specimens taken on this occasion indicated that cryptosporidial infection ended abruptly at the gastroduodenal junction. Electron microscopy revealed numerous Cryptosporidia at various stages of development. They were similar to those described in previous reports [5,‘i’]. Maturing trophozoites, schizonts, merozoites and early macrogametes were identified (Figure 3). They were embedded in microvilli and attached to enterocytes, thereby pushing microvilli away. The enterocytes were indented and had widened terminal webs that were devoid of rootlets of microvilli. Each Cryptosporidium was covered by a parasitophorous membrane. Maturing tropho-
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Flgure 5. Schizont (S) and’macrogamete (M). Schizont is surrounded by a parasitophorous membrane with a widened space and has three anucleolar nuclei. Macrogamete has a welldeveloped attachment zone, and granular endoplasmic reticulum, an anucleolar nucleus, a few dense bodies, and peripherally arranged “polysaccharide” granules. Magnification X 13,000.
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dense aggregates of lymphocytes and monocytes. This change was associated with toxoplasma1 organisms and was most intense at the corticomedullary junctions; in this region, it was associated with necrosis of the tubular epithelium of Henle’s loops. The gastric mucosa was dark red and covered by thick plaques of friable brown material. There were extensive areas of intramural necrosis similar to those in the lungs and lymph nodes. The areas of necrosis involved both submucosa and the external muscle coat, and contained many Toxoplasma organisms. Similar zones of necrosis were also encountered in the small and large bowels. The intestinal epithelium was entirely lost as a result of autolysis; hence no cryptosporidial organisms could be identified at autopsy. The right parietal lobe of the cerebrum contained a 1 cm zone of grossly apparent necrosis. All portions of the central nervous system and the posterior pituitary contained many Toxoplasma organisms. There were many microinfarcts containing lumps of Toxoplasma organisms measuring an average of 11 pm. Electron microscopy showed a disrupted cyst wall and many Toxoplasma. These parasites had double plasma membranes, toxonemes, osmiophilic vacuoles, mitochondria and conoids. Other organs, including the bone marrow, thymus, thyroid and parathyroid glands, showed no pathologic changes. The pancreas, adrenal glands and uterus were without major structural alterations except for perivascular collections of lymphocytes and monocytes. In summary, the immediate cause of this patient’s death was an overwhelming dissemination of Toxoplasma manifested by necrotizing encephalitis, lymphadenitis, pneumonitis and intramural gastroenteritis. The underlying cause of death was severe malnutrition due to cryptosporidial infection of the small intestine.
Figure 6. Attachment zone below the comb-like folded membranes showing a dense band and thin band of the parasitophorous membrane separating Cryptosporidium and enterocyte. The dense band is continuous with the outer layer of the parasitophorous membrane. Note the absence of microvtllous rootlets below the attachment zone. Magnification X 58,000. small and large lymphocytes, histiocytes, plasma cells and neutrophils. There was no suggestion of neoplastic change in any node. Groups of small organisms resembling T. gondii were present in the stromal portions of intact nodes and in the subcapsular lymphatic channels. The lungs contained firm, nonaerated oval gray areas surrounded by a distinct zone of hyperemia. These were most numerous in the lower lobes and displayed the same type of necrosis as that found in the lymph nodes. The necrosis centered about the walls of small pulmonary arteries. The adjacent alveolar spaces were filled with fibrin and nuclear fragments. The histiocytes of the lung contained groups of organisms which ultrastructural analysis identified as Toxoplasma. They had double plasma membranes, mitochondria, toxonemes, conoids and osmiophilic vacuoles (Figure 7). This appearance is consistent with previous reports of this organism [8]. Positive immunofluorescent labeling with Toxoplasma antibody was achieved for these pulmonary parasites, according to the method of Huang et al. [9]. The heart was not unusual to the naked eye and weighed 300 g. The muscle fibers of the left ventricle contained aggregates of toxoplasma1 parasites associated with interstitial aggregates of lymphocytes and monocytes. The liver was greatly enlarged (2,075 g) and showed extensive fatty change but no areas of inflammation or necrosis. The spleen weighed 200 g and the pulp contained many histiocytes showing erythrophagocytosis. The kidneys appeared swollen and weighed 200 and 225 g. Their capsular surfaces were smooth but mottled. The interstitial tissue was infiltrated with
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TOXOPLASMOSIS-STEMMERMANN
COMMENT!3 recorded cases of human small intestinal cryptosporidiosis occurred in immunosuppressed hosts.
The previously
Figure 7. Higher-power electron micrograph showing double plasma membranes, mitochondria (M), polar conoids (C) and toxonemes (T). Magnification X 34.000.
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In one patient the infection ended spontaneously when immunosuppression was corrected [3], but an infection in a child with hypogammaglobulinemia did not respond to medication [4]. Our patient had received no medication known to induce immunosuppression, nor could any illness be identified to account for an immunosuppressed state (e.g., Hodgkin’s disease, Sjiigren’s disease, lymphosarcoma). The patient had a brother and a second cousin who died with lymphosarcoma. Although she had a normal white blood cell count, she displayed a conspicuous lymphocytosis (60 to 81 percent] throughout the last six months of her life, and 25 to 50 percent of her lymphocytes were atypical. Numerous bone marrow examinations were performed, yet none gave any evidence of lymphosarcoma or leukemia. The autopsy also failed to establish this diagnosis. The sudden increase in the number of lymphocytes in the last three weeks of life might have been a response to the disseminated toxoplasmosis or an early manifestation of leukemia. There was evidence of both humoral and. cellular immunologic incompetence-depressed levels of complement and immunoglobulins and decreased T cell reactivity, respectively. The former could be accounted for on the basis of malnutrition causing deficient protein synthesis, the latter on the basis.of altered function of transformed T cells. The antibody levels against cytomegalic virus and Epstein-Barr virus indicate that the patient had previously been infected by these viruses. Cytomegalic virus infection is frequently encountered in the immunologically compromised host, yet there was no evidence of active infection by this virus at autopsy. The decrease in the titer of T. gondii antibody probably reflects a decrease in the production of humoral antibody due to malnutrition. Disseminated toxoplasmosis of the type found in this patient is not usually encountered among adults except when they are under treatment with agents causing immunosuppression [lO,ll]. The synchronous occurrence of cryptosporidiosis and dissem-
ET AL
inated toxoplasmosis suggests that these diseases are subject to similar immunologic restraints. The cryptosporidiosis of immunodeficient Arabian foals [6] would appear to be a good model of the disease in human subjects. The immunodeficiency of these animals is an autosomal recessive disease characterized by lack of development of B and T lymphocytes [x]. It resembles the combined immunodeficiency of the Swiss type that is characterized by lymphopenia, rather than lymphocytosis as in our case. Therapeutic immunosuppression also results in combined immunodeficiency. Neither equine cryptosporidiosis nor any of the cases in human subjects, therefore, offers a clue as to the specific immunologic defect favoring infection by this organism. The disseminated necrotizing inflammation discovered at necropsy posed a problem of interpretation at the light microscopic level. Foci of necrosis in t$e lungs, gut wall, lymph nodes and brain contained organisms resembling T. gondii. We were not entirely certain whether an oocystic stage of Cryptosporidium could be ruled out, even though neither cryptosporidial oocysts nor disseminated cryptosporidiosis have been described. Ultrastructural and immunofluorescent studies of the organisms in the lungs and central nervous system, however, clearly identify them as toxoplasma1 in type. They contained mitochondria and polar conoids, neither of which is found in Cryptosporidia [7]. ACKNOWLEDGMENT
We wish to express our gratitude to Dr. W. M. Meyers, Chief, Microbiology Division, Armed Forces Institute of Pathology, for his assistance in the identification of the cryptosporidial organism: to Dr. 1. K. Frenkel, Department of Pathology, University of Kansas Medical Center, for his assistance in the identification of the toxoplasma1 organism, and to Dr. D. A. Stevens, University of Stanford, School of Medicine, Palo Alto, California and Dr. G. S. Visvesvara, C.D.C., Atlanta, Georgia, for their advice and assistance.
REFERENCES 1. Brownstein DG. Strandberg JD. Montoli RJ, Bush M, Former J: Cryptosporidium in snakes with hypertrophic gastritis. Vet Path01 1977; 14: 606. 2. Vetterling JM, Jervis HR. Merrill TG, Sprinz H: Cryptosporidium wrairi from the guinea pig Cavia porcellus with an emendation of the genus. J Protozool1971; 18: 243. 3. Meisel JL, Perera DR; Meligro C, Rubin CE: Overwhelming watery diarrhea associated with a cryptosporidium in an immunosuppressed patient. Gastroenterology 1976; 70: 1156. 4. Nime FA, Burek JD, Page DL, Holscher MA, Yardley JH: Acute enterocolitis in a human being infected with the protozoan crfrptosporidium. Gastroenterology 1976; 79: 592. 5. Lasser KH, Lewin KJ, Ryning Fw: Cryptosporidial enteritis in a patient with congenital hypogammaglobulinemia. Hum Path01 1979; 10: 234. 6. Snyder SP, England JJ, McChesney AE: Cryptosporidiosis
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&i immunodeficient arabian foals. Vet Patbol 1978; 15: 12. 7. Vetterling JM, Takeuchi A, Madden PA: Ultrastructure of Cryptosporidium wrairi from the guinea pig. J Protozool 1971;18: 248. 8. Callaway CS, Walls KW, Hicklin MD: Electron microscopic studies of toxoplasma gondii. Arch Path01 1968; 86: 484. 9. Huan SN, Minassian H, More JD: Application of immuno ff uorescent staining on paraffin sections improve&by try sin digestion. Lab Invest 1976; 35: 383. 10. Fren t: el JK, Nelson BM, Arias-Stella J: Immunosuppression and toxoplasmic encephalitis. Hum Path01 1975: 6: 97. 11. Frenkel JK, Amore M. Larsen W: Immune competence in a patient with Hodgkin’s disease and relapsing toxoplasmosis. Infection 1978; 6: 3. 12. McGuire TC, Banks KL, Davis WC: Alterations of the thymus and other lymphoid tissue in young horses with combined immunodeficiency. Am J Path01 197fi; 84: 39.
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