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Infections of the lower GI tract Joel K. Greenson, M.D. Professor of University of Michigan Medical School Rm 2G332, Box 0054 1500 East Medical Center Drive Ann Arbor, Michigan 48109-0054 734 946 6770 [email protected]

Isospora belli (Cystoisospora):

I. GENERAL

Isospora belli is a protozoal parasite in the class Sporozoea, a group that also includes toxoplasmosis and cryptosporidiosis. While a variety of Isospora species are recognized in animals, the speciies I.belli is limited to man and is the only Isospora reported in man. I.belli does occur in immunocompetent individuals and is endemic in parts of Central and South America, SouthEast Asia, and Africa. Prior to AIDS, US cases were reported as rare outbreaks in institutions, laboratory accidents, WWII veterans returning from Asia, immigrants, and infants. With the advent of AIDS, Isospora has been reported more frequently. In endemic areas, Isospora is a more frequent cause of AIDS diarrhea, being reported in 15% of Haitians from an AIDS clinic and in 10% of patients attending an AIDS clinic in Sao Paulo, Brazil. In the AIDS population in the US, the highest incidence of Isospora is seen in immigrants from Central and South America (up to 7% of such persons), with less than 1% of the more general US population showing this . There is a strong inverse relationship with the occurence of Isospora and Pneumocystis, since PCP prophylaxis is an effective treatment of Isospora.

II. CLINICAL PICTURE

The clinical presentation of I.belli_ is characterized by watery diarrhea, crampy epigastric abdominal pain, weight loss, anorexia, malaise, and flatulence. The diarrhea and abdominal pain may be exacerbated by eating. Fever and leukocytosis are uncommon while fat malabsorption and peripheral eosinophilia are seen frequently. Stool examination commonly shows Charcot-Leyden crystals, but blood and leukocytes are rare. In immunocompetent individuals, the is usually self-limited, however the infection can become chronic, with a patient reporting 20 years of diarrhea (following military service in North Africa). Deaths have been reported in immunocompetent individuals, from severe electrolyte disturbances, but these cases were from before the most effective antibiotic regimen was recognized. On the other hand, in the immunosuppressed host, the disease is usually a chronic, intermittent illness. And in the 2

immunosuppressed, Isospora has been noted in extraintestinal sites, including as a cause of acalculous cholecystitis and has been noted at in mesenteric lymph nodes, liver and spleen. There is effective therapy for I.belli_, namely trimethoprim-sulfamethoxazole (TMP-SMX), which is usually given for one week. Immunosuppressed patients have a high frequency of recurrence (~50%), but respond readily to retreatment. To prevent relapse, patients with AIDS have been maintained on a lower dose of TMP-SMX or other drug combiniations.

III. DIAGNOSIS BY BIOPSY

In all reported cases, the small bowel morphology is abnormal with club-shaped, blunted villi to absent villi (rarely), and increased chronic in the lamina propria with prominent eosinophils. Various stages of the lifecycle of this parasite can be seen in the small bowel epithelium, with asexual forms predominating. The most readily identified asexual form is the merozooite which matures from clusters of small crescent-shaped structures to single, larger uninucleate banana-shaped forms. These asexual forms are visible on H&E stains within the cytoplasm of the enterocytes of the villi, and range from slightly smaller to larger than the enterocyte nuclei. The sexual forms (microgametes and macrogametes) are less numerous and are best seen with an overstained or Colophonium Giemsa as rounded structures with tiny dark spots in their cytoplasm.

IV. DIAGNOSIS BY STOOL EXAMINATION

Oocysts of I.belli are elliptical and large, measuring 20 to 30 microns by 10 to 20 microns, and are best seen by modified acid fast stain in concentrated stool. In freshly passed stool, immature oocysts are usually found, which contain a single, round granular zygote. Storage of the specimen in potassium dichromate at room temperature for 24 hours promotes sporulation. During sporulation, the zygote divides into 2 sporoblasts which mature into sporocysts containing 4 crescent- shaped sporozoites. Unfortunately, the oocysts may not be numerous during the initial and severe stages of the illnes when asexual multiplication predominates. Thus, diagnosis may require multiple stool examinations and possibley duodenal biopsy.

REFERENCES

Benator DA, French AL, Beaudet LM, Levy CS, Orenstein JM. Isospora belli infection associated with acalculous cholecystitis in a patient with AIDS. Ann Intern Med 121(9):663-664, 1994.

Comin CE, Santucci M. Submicroscopic profile of isospora belli enteritis in a patient with acquired immune deficiency syndrome. Ultrastruct Pathol 18:473- 482, 1994.

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DeHovitz JA, Pape JW, Boncy M, Johnson Jr., WD. Clinical manifestations and therapy of Isospora belli infection in patients with the acquired immunodeficiency syndrome. N Engl J Med 315:87-90, 1986.

Gellin BG, Soave R. Coccidian in AIDS: Toxoplasmosis, Cryptosporidiosis, and Isosporiasis. Medical Clinics of North America 76(1):205- 235, 1992.

Goodgame RW. Understanding intestinal spore-forming protozoa: Cryptosporidia, Microsporidia, Isospora, and Cyclospora. Ann Intern Med 124:429-441, 1996

Cyclospora cayetanensis:

General Comments Cyclospora cayetanensis was first recognized as a human pathogen by Ashford, who reported its occurrence in Papua New Guinea in 1979. This protozoal pathogen was further characterized in the mid-1990s following several outbreaks of diarrheal illness in North America and Europe. Outbreaks in developed countries have been linked to waterborne sources and foods imported from developing countries, such as raspberries and basil, where its transmission is probably due to contaminated water supplies and poor sanitation. Recent outbreak in the Midwest during the summer of 2013 sickened over 60 people and was thought to be due to contaminated lettuce. Children in endemic countries are five times more likely to be infected than adults. Risk factors for infection include drinking or swimming in contaminated water sources, direct contact with soil, and diets rich in raw, or poorly cooked, bivalves. Humans are the only known hosts for Cyclospora cayetanensis.

Clinical Features and Treatment Cyclospora cayetanensis infection usually produces symptoms within 2 weeks of exposure to the pathogen. Gastrointestinal symptoms include loose or watery diarrhea, nausea, vomiting, cramps, flatulence, bloating, and weight loss, but most patients also have systemic manifestations, such as fever, chills, myalgias, and fatigue. Cyclospora cayetanensis may infect either immunocompetent individuals, in whom it produces a self-limited diarrheal illness, or immunodeficient patients, who may suffer life-threatening or protracted symptoms. Trimethoprim- sulfamethoxazole is the standard treatment for cyclosporiasis, and symptoms usually begin to resolve within 1-2 days of treatment initiation. Immunocompetent patients receive a one-week course of twice-daily oral therapy, whereas those with immunodeficiency receive oral doses four times daily for a 10-day period. Prolonged infection may result in biliary disease, acalculous cholecystitis, Guillain- Barr syndrome, or reactive arthritis

Diagnosis and Pathologic Features Cyclospora cayetanensis may be diagnosed using several methods, including PCR-based assays for C. cayetanensis DNA. Oocysts may be observed in stool 4

samples or intestinal aspirates from infected patients. They are non-refractile and show variable staining with a modified acid-fast stain, which is most intense when performed on fresh specimens. The cysts also show bright autofluorescence under ultraviolet illumination. Mucosal biopsy samples from the jejunum and distal duodenum show variable degrees of villous shortening and crypt in association with patchy, usually mild, increased inflammation in the lamina propria and crypt epithelium. The organisms are readily apparent on routine hematoxylin and eosin stained sections, and all of the intracellular stages of the life cycle may be identified. The asexual stages of the life cycle are distinct: first generation meronts contain numerous merozoites ranging from 3-4 m m, whereas second generation meronts contain four larger (12-15 m m) merozoites arranged in parallel arrays. Supranuclear parasitophorous vacuoles are often numerous in the apical compartment of the epithelial cells and contain either sporozoites or mature, free merozoites.

Differential Diagnosis The oocysts of Cyclospora cayetanensis are morphologically similar to those of Cryptosporidium parvum and may cause diagnostic confusion when evaluating stool samples. However, the oocysts of the former are much larger (8-10 mm) than those of the latter (4-5 mm), and the appearances of these pathogens in mucosal biopsy samples are entirely different. Cyclospora are deep-seated intracellular organisms that show various stages of sexual and asexual reproduction, whereas Cryptosporidia are uniform spherical structures that adhere to the luminal surface of the intestinal epithelium, but do not reside within the cytoplasm. The parasitophorous vacuoles of Cyclospora cayetanensis may also simulate the appearance of Isospora belli in tissue sections. However, I. belli cysts are elliptoid, elongated or crescentic in shape, and are much larger (20-33 x 10-19 m m) than those of C. cayetanensis. Thus, the distinction between these parasites is straightforward when one combines morphologic assessment of mucosal biopsy samples and stool studies.

References:

1. Ashford RW: Occurrence of an undescribed coccidian in man in Papua New Guinea. Ann Trop Med Parasitol 1979, 73:497-500

2. Shields JM, Olson BH: Cyclospora cayetanensis: a review of an emerging parasitic coccidian. Int J Parasitol 2003, 33:371-91

3. Sun T, Ilardi CF, Asnis D, et al.: Light and electron microscopic identification of Cyclospora species in the small intestine. Evidence of the presence of asexual life cycle in human host. Am J Clin Pathol 1996, 105:216-20

4. Herwaldt BL: Cyclospora cayetanensis: a review, focusing on the outbreaks of cyclosporiasis in the 1990s. Clin Infect Dis 2000, 31:1040-57

Giardia lamblia: 5

Giardia lamblia was the first protozoa discovered in the human intestine, as Leeuwenhoek describe the organism in his own stools in1681.Giardiasis is reportedly the most prevalent gut parasite in the United States and Britain. While the prevalence of giardiasis has been quoted as 7.4% in the U.S., it may be as high as 50% in some underdeveloped nations. A study of American travelers to Leningrad over a four year period found that 23% were infected with the organism. While many patients with giardia are asymptomatic, a wide variety of gastrointestinal symptoms may occur. Some patients report acute onset diarrhea with greasy fouI-smelling non-bloody stools while others report abdominal distension/bloating, pain, and flatulence. Weight loss and malabsorption may occur and the disease may persist for months if unrecognized/untreated. The organism is often transmitted by contaminated drinking water (fecal/oral), however, intimate contact may also spread the disease as evidenced by the high prevalence of infection in "gay bowel disease." Both endemic and epidemic infections can be seen. The organism is ingested as a cyst present in contaminated food or drinking water. In the stomach, duodenum, and upper small intestine, the parasite transforms into a trophozoite. In the lower small intestine and colon, the organisms form back into cysts which are passed in the stool A number studies have found that children and patients with hypogammaglobulinemia, agammaglobulinemia, IgA deficiency, and/or achlorhydria have a higher incidence of infection with giardia. However, a humoral immunodeficiency state is not necessary for infection. Immunosupressed patients are more likely to have long-term infections with chronic diarrhea and malabsorption, while immunocompetent patients are more likely to have an acute self-limited diarrheal illness or become asymptomatic carriers. While the diagnosis of giardiasisis best made on ova and parasite exams or via a duodenal fluid aspirate, identification of the trophozoite on routine H & E stained sections of the proximal small bowel is often possible. The trophs are pear-shaped and exhibit two nuclei from a ventral view. From a lateral view, the trophs have a sickled shape. In some cases, large numbers of the organisms can be found clumped together in the intravillous spaces, while in others only one or two parasites will be evident in serial sections. Some trophs may be seen attached to the epithelial surface and there are reports of tissue invasion by the organism. On H&E stained sections, the trophs often have a smudgy blue-gray appearance which can easily be overlooked as extravesated mucus or intraluminal debris. By focusing up and down, one may appreciate the two nuclei of the troph peering back through the microscope like eyes on a face. A trichrome, giemsa, PTAH or PAS stain may help bring out the detail of the organisms. In general, it is a good idea not to diagnose giardiasis unless you can see the organisms’ nuclei. The histologic changes seen in the small intestinal mucosa are quite varied. Many patients have completely normal appearing small bowel biopsies, while some have minor non-specific villous blunting and a modest increase in lamina propria mononuclear cells. Other patients have had more substantial 6 pathologic changes, including cases of near total villus reminiscent of untreated sprue. Patients with hypogammaglobulinemia often have the most profound histologic changes, having been referred to as “hypogammaglobulinemic sprue”. Fortunately, these patients’ biopsies often show significant improvement following the eradication of Giardia.

REFERENCES (Giardiasis):

1. Moore GT,Cross M, McGuire D, Mollohan CS, Gleason NN, Healy G R, Newton LH. Epidemic Giardiasis At A Ski Resort. The New England Journal of Medicine 281:402-407,1969. 2. Yardley J H.: Pathology of Chronic Gastritis and Duodenitis. In Goldman H, Appelman HD, Kaufman N,(eds.):Gastrointestinal Pathology. Willams and Wilkins, Baltimore, 1 990, pp 69-1 43. 3 Abrams G D.:Infectious Disorders of the Intestines. In Ming SC, Goldman H,(eds.): Pathology of the Gastrointestinal Tract, W.B. Saunders Co. Philadelphia 1992,pp621-642.

CRYPTOSPORIDIOSIS

1. These are coccidial organisms related to Isospora,Toxoplasma and (remotely) Plasmodia. More common in animals than man, but now known that it is a zoonosis, causing short-term diarrheal illness in immunocompetent humans, persistent and even lethal intestinal illness in immunodeficient and AIDS patients. Can be epidemic.

2. Organisms occur as numerous round, small (2-4 um) basophilic bodies lying within the brush border, especially upper villi. However, EM study demonstrates that they are actually intracytoplasmic, as with coccidia and related protozoans.

3. Cryptosporidia can infect stomach and colon as well as small intestine.. Also reported in bronchi, pancreatic ducts and biliary tree.

4. Diagnosis is usually made from tissue or smear. Diagnosis from stool requires recognition of oocyst form. Acid-fast stain is best for demonstrating oocysts.

References:

1. Current, W.L., Reese, N.C.,Ernst,J.V.,Bailey, W.S.,Heyman, M.B., and Weinstein, W. M. Human cryptosporidiosis in immunocompetent andImmunodeficient persons. Studies of an outbreak and experimental transmission. New England J. Med. 308:1252-1257,1983. 7

2. Garcia, L.S., Brucker, D.A., Brewer,T.C.,and Shimizn, R.Y.: Techniques for the recovery and identification of cryptosporidium oocysts from stool specimens. J. Clin. Microbiol. 18:1 58, 1983 3. Meisel, J.L., Perera, D.R., Meligro, C. and Rubin, C.E.: Overwhelming watery diarrhea associated with cryptosporidium in an immunosuppressed patient. Gastroenterology 70:1156-1160,1978 4. MacKenzie WR, Hoxie NJ, Proctor ME, et al. A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply. N Engl J M ed 1 994; 331:1 61-7. (A first-rate epidemiologic study.)

MICROSPORIDIOSIS

BACKGROUND. The phylum Microspora consists of primitive protozoa that are characterized by the production of unique spores. These spores contain a coiled hollow tube (polar tubule) that can be extruded and through which infective material (sporoplasm) can be injected into host cells. Since the main hosts for these organisms are arthropods and fish, these protozoa received scant medical attention until brought to light in the study of diarrhea in AIDS patients. Although there are over a hundred genera and about a thousand species in this phylum, only a handful of species have thus far been detected in man, and these are collectively designated by the non-taxonomic term microsporidiosis.

The 2 major microsporidia seen in the gut are Enterocytozoon bieneusi and Encephalitozoonintestinalis(formerly Septata intestinalis). In conventional H&E stained biopsies, these organisms are quite difficult to detect due to their minute size and poor staining qualities. Formerly, diagnosis rested o n electron microscopy of tissue samples, but diagnosis is now being rendered more frequently by light microscopy (special stains) on biopsies, nasal smears, stool, sputum, and urine samples.

ENTEROCYTOZOON BIENEUSI. Ebieneusi was first described in 1985, and is still the major microsporidial infection in man, particularly in AIDS patients with diarrhea. Prospective studies of HIV-associated chronic diarrhea indicate a prevalence of 9 to 1 6% in patients evaluated by stool studies to detect microsporidia. Recently, this organism was reported to have caused a self-limited traveler's diarrhea in a non-immunocompromised individual, and it is likely that this occurs more frequently but is not recognized. While originally described as limited to small bowel, this organism has subsequently been detected in the biliary system. Clinical Symptoms. The most commonly reported symptoms with Enterocytozoon bieneusi infections are chronic diarrhea, anorexia, and weight loss, and less frequently - abdominal pain, nausea, vomiting and fever. These latter symptoms are more common in patients with concomitant biliary disease which may produce a clinical picture of cholangitis or cholecystitis. Not all patients develop diarrhea and the exact relationship between infection and clinical illness has 8

been disputed. Those individuals with chronic debilitating disease usually have advanced AIDS with very low CD4 counts. Treatment is primarily supportive as there is no effective drug to treat this particular infection.

Diagnosis by Electron Microscopy. Two phases of the lifecycle of E.bieneusi can be identified by EM, a proliferative phase (merogony) and a spore-forming phase (sporogony). In the proliferative phase, the organisms are small rounded objects with 1 to 6 nuclei, and are more electron-lucent than the surrounding cytoplasm of th e surrounding host cell. Very few structures are present in the cytoplasm of the organisms with empty clefts (electron lucent inclusions) being the major feature. The herald of the spore-forming phase is the presence of stacks of electron dense discs. At later stages, the discs aggregate end-to-end to form the longer, curved profiles of polar tubes. During sporogony, the nuclei continue to divide, resulting in large organisms with up to twelve nuclei with each nucleus surrounded by several coils of polar tube. Finally, these large multinucleated forms break u p into immature spores (sporoblasts) which then develop into mature spores. Mature spores are extremely electron dense, have a single nucleus, and an extrusion appartus consisting of several coils of polar tube, and anchoring disc, and a polarplast. The coils of polar tube are in 2 rows. Both phases of the life cycle are localized to the apical portion of enterocytes(supranuclear), and multiple or all stages of the life cycle can be identified in a single cell. All phases of the life cycle lie free within the host cytoplasm, with no limiting vacuole or membrane. Frequently, E bieneusi(which lack their own mitochondria) are closely apposed to host mitochondria, presumable siphoning off energy.

Diagnosis by Light Microscopy. Although E bieneusi has been reported in normal small bowel mucosa, it is more frequently detected in biopsies showing some histologic abnormalities. The highest concentrations of organisms tend to be in villous tips which have a degree of blunting, focally increased intraepithelial lymphocytes, and some shedding of enterocytes; and it is in these abnormal areas that the hunt for organisms should be concentrated. On conventional H&E stains, the organisms appear as small pale blue structures in the supranuclear cytoplasm which may be surrounded by a halo or indent the nucleus. H&E diagnosis alone is unreliable and needs to be supported by special stains for the spores. The spores are gram positive and are usually clustered together in the apical cytoplasm of enterocytes at villus tips. The spores are also highlighted by modified Trichrome stains, Warthin-Starry, and in 1 micron plastic sections by toludine blue or azure II methylene blue. Unfortunately, the proportion of mature spores to multinucleated forms is unpredictable and maybe low. Biopsies of jejunum have a higher diagnostic yield than duodenum or ileum, although the latter sites may be diagnostic.

Diagnosis by Stool Examination. Spores are shed in stool but are difficult to detect due to their small size and resemblance to yeast. Current recommendation for detecting spores of E. bieneusi are using fixed smears and screening with an optical brightener such as calcofluor white (that binds the chitin in the spore wall) and then confirming with a modified trichrome stain. By the modified trichrome stain, the spores are seen as small pink ovals, approximately 1.5 x 0.5 microns, 9

which have a polar cleared zone, giving the appearance of a band around the middle of the spore. Although they also stain pink, small yeast such as Candida are rounder and more homogeneous with no internal structures. Bacteria and most fecal debris stain only with the counterstain.

ENCEPHALITIZOON INTESTINALIS. A second microsporidian that involves the GI tract, E. intestinalis(formerly Septata intestinalis), was first described in 1 992. But in addition to infecting the small bowel and biliary system, E. intestinalis can disseminate to involve the respiratory system, urinary system, and conjunctiva. Although E. intestinalis is less frequent than E. bieneusi, it is important to recognize since effective therapy is available.

Clinical Symptoms. The primary clinical complaints are severe watery diarrhea and weight loss followed by a sclerosing cholangitis-like picture with RUQ pain and fever, with ultrasound showing a diseased biliary system. Other symptoms include sinusitis, bronchitis, and conjunctivitis. While spores are frequently seen in urine, urogenital symptoms are surprisingly minimal and creatinine is usually normal. Clinical symptoms respond rapidly and dramatically to albendazole therapy,but relapses can occur following cessation of therapy.

Diagnosis by EM. The various proliferative and spore-forming stages of Eintestinalis occur together within a cluster, with the late stages centrally located and the earlier stages more peripheral in the clusters. The early proliferative phase consists of relatively simple structures with 1 to 4 nuclei, and are embedded in a fine fibrillar network secreted by the organism. The sporogenic phase is heralded by the accumulation of electron-dense material along the cell surface giving it a scalloped appearance that then gives way to a thick membrane, which eventually becomes the exospore coat. Cells with the thick membrane continue to undergo nuclear and cytoplasmic division and when the last cell division is completed, spore-specific organelles appear including the polar tube. As the spores mature, an electron-lucent space develops around each spore within the fibrillar network, giving the appearance that the spores are suspended in a honeycomb. The mature spores measure 2 x1.5 microns, have a single nucleus, and 5 turns of the polar tube in a single row.

Diagnosis by Light Microscopy. Small bowel biopsies findings are similar to those E. bieneusi, but differences do exist. Again, organisms appear as indistinct, small bluish structures in the apical cytoplasm of enterocytes. The organisms are somewhat more refractile (perhaps due to heavier spore production). Gram stain will highlight the spores in the enterocytes, but in E. intestinalis infection will also show spores in the macrophages, fibroblasts and endothelial cells of the lamina propria. Thus gram staining can help speciate the 2 major microsporidia seen in the gut. In addition to being found in the small bowel, the organisms can be seen in the biliary system, as well as in urine, nasal smears, and sputum (free and in cells). The use of exfoliative and aspirated cytologic methods to detect human microsporidiosis is assuming increasing importance with a gram or Weber chromotope stain done to identify spores.

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Diagnosis by Stool Exam. Screening with calcuflour white and confirmation with a modified trichrome stain is the current suggested method for detecting the spores of E. intestinalis. As in E. bieneusi,the spores are oval and by trichrome stain pink with a stripe, but the spores of E. intestinalis are larger, measuring approximately 2.5 x 1.5 microns.

Comparison of Microsporidia in the Gut:

E. bieneusi E. intestinalis Disseminated disease No Yes Major sites of infection Small bowel Small bowel Biliary system Biliary system Respiratory (sinuses, bronchi) Genitourinary (kidney, bladder Small bowel bx Organisms in apical Organisms in apical enterocytes enterocytes and lamina Few Gram + spores propria cells Many Gram + spores Spores 1-1.5x.5 microns 2-2.5x1.5 microns Double row polar tubules Single row polar tubules E M Electron dense discs "Septate" parasitophorous vacuoles Effective therapy No Yes

REFERENCES (Microsporidiosis):

1. Cali A, Owen R L. Microsporidiosis In: Balows A, ed., Laboratory diagnosis of infectious disease: Principles and practices. New York. Springer-Verlag.1988. Vol 1,Chap 98,Pgs 929-950. 2. Cali A, Owen RL. Intracellular development of Enterocytozoon, a unique microsporidian found in the intestine of AIDS patients. J Protozool 37:145- 155, 1990. 3. DesportesI,LeCharpentierY,Galian A, Bernard F,Cochand-Priollet B, Lavergne A, Ravisse P, ModiglianiR: Occurrence of a new microsporidian Enterocytozoon bieneusi occuring in the enterocytes of a human patient with AIDS. J Protozoo132:250-254,1985. 4. Orenstein J M, Chiang J, Stein berg W, Smith PD, Rotterdam H, Kotler DP. Intestinal microsporidiosis as a cause of diarrhea in human immunodeficiency virus-infected patients. Hum Pathol 21:475-481, 1 990. 5. Greenson JK,Belitsos PC, Yardley JH,BartlettJG. AIDS enteropathy-occult enteric infections and duodenal mucosalalterationsin chronic diarrhea. Ann Intern Med114:366-372,1991. 6. Weber R, Bryan RT, Owen RL, Wilcox CM, Gorelkin L,Visvesvara GS. Enteric Opportunistic lnfections Working Group. Improved light-microscopic 11

detection of microsporidia spores in stool and duodenal aspirates. New EngI J Med 1992;326:161-6.

TROPICAL SPRUE (POST-INFECTIVE TROPICAL MALABSORPTION)

1. Definition (Baker and Mathan):"Intestinal malabsorption of unknown etiology, occurring among residents in, or visitors to, the tropics". Despite the fact that a single etiologic agent has not been identified, there is much evidence that an infection initiates and sustains tropical sprue: a) It occurs in certain specific geographic areas (eg, West Indies, Indian subcontinent) and enteric infections are common in these locations. b) In some areas it is epidemic. c) Aerobic enterobacteria colonize the patient's small intestine and these may be toxin producing. (Note that this differs from the stasis syndrome in which anaerobic bacterial overgrowth is central (see below)). d) Recovery after treating tropical sprue with broad-spectrum antibiotics is usually rapid and dramatic. e) Some have postulated that a Protozoan infection such as cyclospora may play a role.

The important role played by infection in tropical sprue has led to the alternative designation "Post-infective tropical malabsorption". 2. Other factors such as epithelial damage may also contribute to the condition. Along with diarrhea and malabsorption, folate, and sometimes B-12 deficiencies are commonly present. In severe tropical sprue there can be resulting diminution in epithelial mitosis accompanied by nuclear enlargement - changes that are the epithelial counterpart to maturational derangements in the marrow and macrocytic anemia. Genetic or ethnic predisposition has also been suggested.

3. The mucosal lesion in tropical sprue is of "nonspecific"type with epithelial blunting, chronic inflammation, etc. A completely flat biopsy like that often seen in celiac disease is rare in tropical sprue, but epithelial dysfunction, as in celiac disease, is central to . Unlike celiac disease, in which mucosal changes are greatest in the proximal small bowel, lesions in the ileum are as prominent as those in the jejunum in tropical sprue. This fits well with the resulting secondary B-12 and folate deficiency states (which are not common in celiac disease).

STASIS (Bacterial overgrowth)

1. Primary lesion in scleroderma is in muscularis propria. Smooth muscle, especially inner (circular) coat is replaced by fibrosis, causing sacular dilatation and diverticulum formation. This is a good example of the stasis or blind loop syndrome, which can occur in many circumstances of altered structure and motility (Table 3). See Fig 2 for pathogenesis. Scleroderma of the small intestine must be distinguished from other forms of muscle degeneration.

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2. Although there is evidence that epithelial injury contributes to the malabsorption, the mucosa typically looks normal or is altered only secondarily and nonspecifically. Hence mucosal biopsy will not provide definitive diagnosis in scleroderma and other neuromuscular causes of the stasis syndrome.

REFERENCES (Tropical Sprue and bacterial overgrowth):

1. Baker, 2.7. and M ath an, V.I.: Syndrome of tropical sprue in India. Am. J. Clin. Nutr. 21:984, 1968. 2. Schenk, E.A., Samloff, I .M. and Klipstein, F.A.: Morphologic characteristics of jejunal biopsy in celiac disease and tropical sprue. Am. J. Path. 47:765,1965. 3. Swanson, V.L.and Thomassen,R.W.:Pathology of the jejunal mucosa in tropical sprue. Am. J. Path. 46:511,1965. 4. Swanson, V.L., Wheby, M.S. and Bayless, T.M.:Morphologic effects of folic acid and vitamin B-12 on the jejunal lesion of tropical sprue. Am. J. Path. 49: 167, 1966. 5. Trier JS, Donnelly SM. Case records of the Massachusettes General Hospital. New EngI J Med 322:1067-1075,1990. [CPC, good case, good up-to-date review and reference source]. 6. Wheby,M.S.,Swanson,V.L.and Bayless, T.M.:Comparison of ileal and jejunal biopsies in tropical sprue. Am. J. Clin. N utr. 24:11 7-1 23,1 971. (Sheds additional light on distribution of mucosal lesion in tropical sprue and compares with celiac disease). 7. Cook GC. Aetiology and pathogenesis of postinfective tropical malabsorption (tropical sprue).Lancet1:721-723,1984. 8. Cook GC. Postinfective malabsorption (includes tropical sprue). In: BouchierAD.Alan RN, Hodgson JF, Deighley M RB, eds.: Gastroenterology. Clinical practice and science. Philadelphia, Saunders, 1993. Pgs.522-537. (In addition to containing an excellent discussion of tropical sprue, this article gives overall coverage to chronic diarrhea of infectious origin.) 9. Cook GC. "Tropical sprue":Some early investigators favored an infective cause, but was a coccidian protozoan involved? Gut; 40:428-429. 10. Ament, M.E.,Shimoda,S.S.,Saunders,D.P.and Rubin, C.E.1972. Pathogenesis of steatorrhea in three cases of small intestinal stasis syndrome. Gastroenterology 63:728. 11. Tursi A, Brandimarte G, Giorgetti G. High prevalence of small intestinal bacterial overgrowth in celiac patients with persistence of gastrointestinal symptoms after gluten withdrawal. Am J Gastroenterology, 98(4):839-843, 2003.

WHIPPLE'S DISEASE

In 1907, George Whipple described a 36 year old patient with a “hitherto undescribed disease characterized anatomically by deposits of fat and fatty acids 13

in the intestinal and mesenteric lymphatic tissue.” He named the disease intestinal lipodystrophy, while today we recognize this entity as Whipple’s disease. Whipple’s disease patients tend to be middle-aged white males. Patients typically present with diarrhea, malabsorption, weight loss and abdominal pain, however, systemic and neurologic symptoms may overshadow the GI complaints. Some patients present with a long-standing fevers of unknown origin (FUO), lymphadenopathy, and arthralgias, which may be misdiagnosed as sarcoidosis (biopsies from non-GI sites may show small granulomas). Other patients may manifest only CNS type symptoms. Increased skin pigmentation and protein- losing enteropathy may also develop. Endoscopically, the small bowel in Whipple’s disease may show thickened folds with yellow-white granular deposits.

It should be remembered that this is a rare disease and that there have been more papers written on Whipple’s disease than patients with Whipple’s disease. Nevertheless, patients who present with both CNS and GI disease often require a small bowel biopsy to rule out Whipple’s.

While George Whipple described the presence of rod-shaped bacteria in the lymphoid tissues of his original report and commented that they might play an etiologic role, the disease was thought to be a lipid storage disorder until the bacteria were rediscovered in the early 1960’s with the advent of electron microscopy. Previous attempts at culturing the bacteria has led to varied and disappointing results, as many different bacteria have grown-out, including various streptococcal species. In 1992, however, Relman and colleagues identified the “Whipple bacillus” as Tropheryma whippelii using PCR techniques. This was followed in 2002 by successful culture of the organism inside cultered human macrophages.

Several studies have shown that Whipple’s patients have an underlying defect in their T-lymphocytes and that their cutaneous response to antigenic stimulation is also decreased. While some postulate that these defects are secondary to malabsorption, no defects in humoral immunity have been found, and correction of these defects has not occurred after successful antibiotic therapy. Also, there is an association with HLA-B27 and there are several reports of Whipple’s disease patients with additional GI pathogens such as giardia. All of this data suggests that an underlying immune deficit is present in these patients.

Histopathology: The classic finding in Whipples disease is the presence of numerous PAS positive diastase resistant macrophages filling the lamina propria of the small intestine. The villi are often blunted, rounded, and/or club-shaped and contain macrophages, occasional neutrophils, and lipid droplets. Dilated lacteals may also be present. The macrophages have pink foamy cytoplasm on H&E stained sections. A PAS stain will show that the macrophage cytoplasm is packed with granular inclusions, while the extracellular areas may show PAS positive rod- shaped objects which correspond to intact bacteria. A silver stain may be used to highlight these intact organisms. Ultrastructural studies have found that the macrophages contain intact bacteria as well as digested remnants of bacteria 14 present in phagosomes. It is thought that the inner membrane of the bacterial cell wall is the PAS positive material left behind after the rest of the bacteria is degraded. Intact bacteria have also been found within epithelial cells, endothelial cells, lymphocytes, plasma cells, mast cells, and fibroblasts. After antibiotic therapy, the PAS positive macrophages may remain within the lamina propria for years, however, intact bacteria usually disappear shortly after the onset of antimicrobial therapy. Treatment generally takes at least 1 year to insure adequate coverage of CNS disease.

As Whipple’s disease is a systemic illness, the pathologist may encounter other tissues which contain PAS positive macrophages. Lymph nodes will contain small granulomatous clusters of foamy macrophages, as will the liver, heart, brain, eyes, and the colorectum. In some cases these extra-intestinal sites will be involved years before the onset of diarrhea. The diagnosis is much harder to make, as the presence of small clusters of PAS positive cells are not nearly as specific in a lymph node or liver biopsy as they are in the small bowel. In addition, some cases will not have any PAS positive material present in extra-intestinal granulomas, such that the disease mimics sarcoidosis. Colon biopsies are particularly difficult to interpret, as muciphages can look nearly identical to the foamy macrophages of Whipple’s disease. Most muciphages of the colorectum will stain positively with alcian blue (pH 2.5), while Whipple’s cells will not. Similarly, one may encounter lipochrome containing macrophages in the small bowel that resemble the foamy macrophages of Whipple’s disease, although when present, they are usually not enough cells present to mimic untreated Whipple’s disease. The most important histologic look alike to Whipple’s disease in the small bowel is Mycobacterium avium-intracellulare (MAI) infection in AIDS patients. MAI may present in much the same way as Whipple’s disease, with diarrhea, abdominal pain, weight loss, and neurologic abnormalities (due to opportunistic CNS infections). On H&E stained sections the macrophages of MAI infection have more of a blue-gray hue than the pink cytoplasm seen in Whipple’s cells. As both will stain positively with PAS stains, one must do an acid-fast stain to ultimately differentiate these two entities. It is interesting to note that both Whipple’s disease and MAI infection of the gut arise within the setting of T-cell dysfunction, possibly due to ineffective T-cell/macrophage interaction.

One final problem encountered in the biopsy diagnosis of Whipple’s disease is sampling error. A few reports of a submucosal varient of Whipple’s disease exist, and I have seen such a case myself. The lamina propria looked completely normal, yet the submucosa contained PAS-positive macrophages which I would have readily ignored if the contributing pathologist had not ordered the PAS stain. Presumably patients with submucosal disease have been treated with antibiotics for something else which treated the GI disease, but did not eradicate the CNS infection.

Occasionally, the distribution of foamy macrophages in the lamina propria is patchy, such that one or two biopsies may appear essentially normal. Hence it is better to take multiple biopsies, particularly if the endoscopic findings are unremarkable. 15

Biopsy techniques and ancillary studies: When dealing with the small intestine, the most important thing to communicate to our clinical colleagues is where to take biopsies from. While the duodenal bulb and the terminal ileum are the two most accesible sites in the small bowel, they are also the least helpful to biopsy. Biopsies from the proximal duodenum often have evidence of peptic damage which can confound evaluation of villus architecture. Biopsies from the terminal ileum often have peyers patches which again distort the villus architecure. In addition, many pathologists over- interpret these peyers patches as lymphoid hyperplasia or chronic ileitis. For these reasons, the distal duodenum and/or proximal jejunum are preferable biopsy sites.

In Whipples’ disease it is advisable to take multiple biopsies, as the classic PAS positive macrophages may have a spoty distribution. Endoscopists should be encouraged to biopsy any yellow-white granular areas. In addition, biopsies should be deep enough to contain some submucosa so that the submucosal form of the disease can be identified if present.

As mentioned above, Follow-up biopsies often show the persistance of the foamy macrophages for a long time after treatment, however, the identification of intact bacteria indicates that treatment has not yet been complete. Silver stains and electron microscopy may help identify these intact bacteria, but it is uncertain whether this is necessary from a clinical point of few, as most patients require at least one year of therapy to insure eradication of CNS disease.

A polyclonal immunostain has been developed which was used to stain Whipples’ original cases from Johns Hopkins. While this has shown promise in research articles, I have yet to see data on any commercially available antibodies.

PCR for the identification of Whipples disease can be done on paraffin embedded intestinal biopsies. While this has been shown to be a fairly sensitive and specific test, it does not appear to be useful in monitoring adequacy of treatment. We recently had a case of histologically normal small bowel biopsy that was sent for PCR and came back positive. This prompted a brain biopsy that also appeared histologically normal but was PCR positive. What this means is anybodies guess, but since the Whipples bacillus is thought by some to be part of the normal flora of the colon, I am skeptical that a positive PCR assay in the absence of pathologic changes indicates true infection.

References:

1. Whipple GH. A hitherto undescribed disease characterized anatomically by deposits of fat and fatty acids in the intestinal and mesenteric lymphatic tissues. Johns Hopkins Hosp Bull 18: 382-391, 1907. 2. Cho C, Linscheer WG, Hirschkorn MA, Ashutosh K. Sarcoidlike Granulomas as an Early Manifestation of Whipple’s Disease. Gastroenterology 87:941-7, 1984. 16

3. Wilcox GM, Tronic BS, Schecter DJ, Arron MJ, Righi DF, Weiner NJ. Periodic Acid-Schiff-Negative Granulomatous Lymphadenopathy in Patient with Whipple’s Disease: Localization of the Wipple Bacillus to Noncaseating Granulomas by Electron Microscopy. American Journal of Medicine 83:165-170, 1987. 4. Gillin JS, Urmacher C, West R, Shike M. Disseminated Mycobacterium Avium-Intracellular Infection in Acquired Immunodeficiency Syndrome Mimicking Whipple’s Disease. Gastroenterology 85:2287-91, 1983. 5. Relman DA, Schmidt TM, MacDermott RP, Falkow S. Identification of the Uncultured Bacillus of Whipple’s Disease. The New England Journal of Medicine 327:293-301, 1992. 6. Morningstar WA. Whipple’s Disease: An Example of the Value of the Electron Microscope in Diagnosis, Follow-up, and Correlation of a Pathologic Process. Hum Path 6:443-454, 1975. 7. Fleming JL, Weisner RH, Shorter RG. Whipple’s Disease: Clinical, Biochemical, and Histopathologic Features and Assessment of Treatment in 29 Patients. Mayo Clinic Proceedings 63:539-551, 1988. 8. Kuhajda FP, Belitsos NJ, Keren DF, Hutchins GM. A Submucosal Variant of Whipple’s Disease. Gastroenterology 82:46-50, 1982. 9. Rodarte JR, Garrison CO, Holley KE, Fontana RS. Whipple’s Disease Simulating Sarcoidosis: A Case With Unique Clinical and Histologic Features. Arch Intern Med 129:479-482, 1972. 10. Gonzalez-Licea A, Yardley JH. Whipple’s Disease in the Rectum: Light and Electron Microscopic Findings. The American Journal of Pathology 52:1191-1206, 1968. 11. Lowsky R, Archer GL, Fyles G, Minden M, Curtis J, Messner H, Atkins H, Patterson B, Willey BM, McGeer A. Brief Report: Diagnosis of Whipple’s Disease By Molecular Analysis of Peripheral Blood. The New England Journal of Medicine 331:1343-1346, 1994. 12. Yardley JH.: Malabsorptive Disorders. In Ming SC, Goldman H,(eds.): Pathology of the Gastrointestinal Tract, W.B. Saunders Co. Philadelphia 1992, pp 725-767. 13. Baisden BL, Lepidi H, Raoult D, Argani P, Yardley JH, Dumler JS. Diagnosis of Whipple disease by immunohistochemical analysis: a sensitive and specific method for the detection of Tropheryma whipplei (the Whipple bacillus) in paraffin-embedded tissue. Am J Clin Pathol 2002;118:742-748. 14. Bentley S, Maiwald M, Murphy LD, et al. Sequence and analysis of the genome of the Whipple´s disease bacterium Tropheryma whipplei. Lancet 361: 637-644, 2003. 15. von Herbay A. Whipple´s Disease Online: (last update). URL http://www. WhipplesDisease.net

STRONGYLOIDES STERCORALIS

Strongyloides stercoralis is an intestinal nematode that is found in the tropics and subtropical / temperate areas. In the US, it may be endemic in the southern 17

Appalachian states, although one typically thinks of Africa, the West Indies, Southeast Asia and South America as more common places to be exposed to the infection. Prevalence rates of up to 4% have been reported in Tennessee, while in undeveloped tropical locations it runs as high as 85%.

The organism has a complex life cycle that often results in autoinfection and chronic infection. Filariform larvae can easily penetrate human skin and typically infect people who go barefoot in fecally contaminated soil. These larvae enter the circulatory system and infiltrated the alveolar parenchyma of the lung. Ultimately they work their way up the bronchial tree and are swallowed. Once they reach the small intestine they mature into adult females. The adult female deposits eggs which hatch into Rhabditiform larvae. These may then be passed into the stool or they may initiate autoinfection by becoming infective filariform larvae. This can lead to low-level longstanding infection (in some cases many decades after initial exposure). The longest lasting case on record is 65 years. In patients that become immunosuppressed the infection can become widely disseminated.

Surgical pathologists may recognize adult female worms, eggs and larvae in the small bowel and occasionally the stomach (most commonly seen in the duodenum). The larval forms may also be seen in the colon. The adult worm is typically seen within the small intestinal crypts. Cross-sections will often show multiple eggs within the worm. The worms’ ovaries stain intensely with hematoxylin and make one think about large viral inclusions. The larval stages are smaller with a diameter of 14-16 microns. They have a thin cuticle and rows of small 1-2 micron nuclei that when seen by themselves can make one worry about a small yeast such as Histoplasma. Up to 30% of infected patients are asymptomatic. While most patients with symptomatic Strongyloidiasis are immunosuppressed, not all of them are. Aside from GI complaints, patients may also have skin lesions, pulmonary disease, renal disease and gram-negative sepsis. Immunosuppressive therapy, diabetes mellitus, HTLV-1 infection, organ transplantation, hematologic malignancy, hypogammaglobulinemia, uremia, malnutrition and chronic alcoholism are risk 18 factors for hyperinfection. One must always be careful starting immunosuppression on a patient that has been in an endemic area (even many decades ago). A classic example is that of Viet Nam war veterans who were exposed back in the 1960’s and 70’s who are asymptomatic, but can develop hyperinfection if immunosuppressed.

Stool testing for Strongyloides is not that sensitive with false negative rates reported between 25 and 46%. Multiple repeat stool exams can raise the sensitivity with one report stating 90% sensitivity if 7 or more stool samples are tested. Examining duodenal fluid aspirates may be the most sensitive technique. Serologic testing (ELISA) is also available.

Treatment is relatively simple and effective (if started in time). The drugs ivermectin, albendazole and thiabendazole have all been used (either alone or in combination.

REFERENCES

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Al Samman M, Haque S, Long JD. Strongyloidiasis colitis: a case report and review of the literature. J Clin Gastroenterol. 1999 Jan;28(1):77-80.

Genta RM, Weesner R, Douce RW, Huitger OC, Walzer PD. Strongyloidiasis in US veterans of the Vietnam and other wars. JAMA 1987;258:49-52.

Genta RM. Global prevalence of strongyloidiasis: critical review with epidemiologic insights into the prevention of disseminated disease. Rev Infect Dis 1989;11:755-67.

Qu Z, Kundu UR, Abadeer RA, Wanger A. Strongyloides colitis is a lethal mimic of ulcerative colitis: the key morphologic differential diagnosis. Hum Pathol. 2009 Apr;40(4):572-7. Epub 2009 Jan 13.

Segarra-Newnham M. Manifestations, diagnosis, and treatment of Strongyloides stercoralis infection. Ann Pharmacother. 2007 Dec;41(12):1992-2001. Epub 2007 19

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Sridhara S, Simon N, Raghuraman U, Crowson N, Aggarwal V. Strongyloides stercoralis pancolitis in an immunocompetent patient. Gastrointest Endosc. 2008 Jul;68(1):196-9. Epub 2008 Mar 7.

Weight SC, Barrie WW. Colonic Strongyloides stercoralis infection masquerading as ulcerative colitis. J R Coll Surg Edinb. 1997 Jun;42(3):202-3.

Thompson BF, Fry LC, Wells CD, Olmos M, Lee DH, Lazenby AJ, Monkemuller KE. The spectrum910. of GI strongyloidiasis: an endoscopic-pathologic study. Gastrointestinal Endoscopy 2004 59(7):906-910.

Gutierrez Y, Bhatia P, Garbadawala ST, Dobson JR, Wallace TM, Carey TE. Strongyloides stercoralis eosinophilic granulomatous enterocolitis. Am J Surg Pathol. 1996 May;20(5):603-12.

Ghoshal UC, Alexender G, Ghoshal U, Tripathi S, Krishnani N. Strongyloides stercoralis infestation in a patient with severe ulcerative colitis. Indian J Med Sci. 2006 Mar;60(3):106-10.

Banerjee D, Deb R, Dar L, Mirdha BR, Pati SK, Thareja S, Falodia S, Ahuja V. High frequency of parasitic and viral stool pathogens in patients with active ulcerative colitis: report from a tropical country. Scand J Gastroenterol. 2009;44(3):325-31. http://www.dpd.cdc.gov/dpdx/HTML/Strongyloidiasis.htm