RELATION OF GIARDIASIS TO ABNORMAL INTESTINAL STRUCTURE AND FUNCTION IN GASTROINTESTINAL IMMUNODEFICIENCY SYNDROMES

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1 GASTROENTEROLOGY Copyright 1972 by The Williams & Wilkins Co. Vol. 62, No.2 Printed in U. S. A. RELATION OF GIARDIASIS TO ABNORMAL INTESTINAL STRUCTURE AND FUNCTION IN GASTROINTESTINAL IMMUNODEFICIENCY SYNDROMES MARVIN E. AMENT, M.D., AND CYRUS E. RUBIN, M.D. Department of Medicine, University of Washington, Seattle, Washington Seven of 8 patients with hypogammaglobulinemia and gastrointestinal symptoms were infected with Giardia Lamblia. This parasite was not detected by multiple stool examinations in 4 of the 7 infected patients but was diagnosed correctly by small intestinal biopsy. Abnormalities of the small intestinal mucosa were found in all 8 patients. Two patients had nodular lymphoid hyperplasia and 3 the flat lesion of hypogammaglobulinemic sprue. The remaining 3 patients had mixed lesions; that is, the lesions varied both in severity of the villus abnormality and in the frequency of lymphoid nodules. 1\11 patients lacked plasma cells in the lamina propria. Because of the variation in the severity of the small intestinal abnormalities within individual patients, multiple small bowel biopsies were needed to evaluate the effects of treatment on mucosal morphology. After eradication of Giardia Lamblia in 7 patients, the abnormalities in villus structure returned towards normal in all. No change occurred after a course of metronidazole (Flagyl) in the 1 patient who had a severe intestinal lesion but no evidence of giardiasis. Four patients with steatorrhea and giardiasis gained weight and their fecal fat excretion returned to normal. Diarrhea disappeared after treatment in the remaining 3 patients. Chronic diarrhea and malabsorption l - 14 as well as various small intestinal abnormalities are common in immunodeficiency syndromes presenting later in life. We Received July 6, Accepted September 21, Address reprint requests to: Dr. Cyrus E. Rubin, Department of Medicine, University of Washington, Seattle, Washington This work was supported by Research Grant 20-9R81 from the Children's Orthopedic Hospital and Medical Center; Research Grant CA from the National Cancer Institute, National Institutes of Health, United States Public Health Service; the Clinical Research Center of the University of Washington (National Institutes of Health Grant RR-37); Gastroenterology Training Grant 5 T01 AM05099 (Dr. Ament); and Career Award CA from the National Institutes of Health, United States Public Health Service (Dr. Rubin). The technical assistance of Linda Esther and Ina Bradner is gratefully acknowledged. 216 will refer to this group of disorders as gastrointestinal immunodeficiency. Gastrointestinal problems are not seen in infantile X-linked agammaglobulinemia. 15 Two distinctive intestinal histological patterns have been described in gastrointestinal immunodeficiency. The first has been named nodular lymphoid hyperplasia 13 because of the myriad of lymphoid nodules within the mucosa. These nodules are evident as multiple small polyps; the architecture of the villi is relatively intact except where distorted by lymphoid nodules. 13, 14 These patients usually have mild or only intermittent gastrointestinal symptoms. The second pattern has been called hypogammaglobulinemic sprue l6 because the mucosa is flat and the intestinal villi have largely disappeared These patients have obvious malabsorption and, in our experi-

2 February 1972 GIARDIASIS IN GASTROINTESTINAL IMMUNODEFICIENCY SYNDROMES 217 ence, do not respond to a gluten-free diet. Plasma cells are almost completely absent within the intestinal lamina propria in each of the above entities. Each of these histological entities probably represents opposite ends of the whole spectrum of gastrointestinal immunodeficiency. However, most patients have a mixed histological picture rather than the obvious pattern at either end of the spectrum. In our experience, and in that of others,6, 8, 10 giardiasis and malabsorption are frequent in all of these patients but it is not clear whether this protozoa causes the intestinal abnormalities, Because Giardia lamblia may cause small intestinal lesions in some patients whose immunoglobulins.are normal,17, 18 we wondered whether it might not also contribute to the various intestinal abnormalities seen in gastrointestinal immunodeficiency, Therefore small intestinal structure and function were evaluated before and after eradication of Giardia Lamblia by metronidazole. Patients Sixteen patients with immunodeficiency syndromes were studied to determine if they had malabsorption and giardiasis. Eight of the 16 were classified according to the World Health Organi..:ation recommendation 19 as infantile X-linked agammaglobulinemia; they were excluded from further study because they were not infected with Giardia lamblia, did not have malabsorption, and had practically normal intestinal mucosa except for absent plasma cells. The remaining 8 patients had gastrointestinal symptoms and intestinal abnormalities and are hereafter classified as having "gastrointestinal immunodeficiency." They were subclassified according to the histopathology of their pretreatment biopsies as benign lymphoid hyperplasia, mixed 'lesions, and hypogammaglobulinemic sprue (table 1). Methods Every patient studied had a least one stool examination for cysts and trophozoites of Giardia lamblia. Most had three or four stool specimens examined. Direct fecal smears in physiological saline and 1% KI and a stool concentration technique were used to examine each specimen.20 These patients were biopsied repeatedly with the infant or adult multipurpose 21 or hydraulic biopsy tubes,22 both before and after metronidazole treatment. All biopsies were taken between the duodenojejunal junction and 1 foot distally. In patient 1 the entire TABLE 1 Clinical data Group Patient Age Sex Diarrhea Duration -y-globulin Prior therapy Immunoglobulina IgG IgM IgA Mixed lesions 1 50 M Watery 14 mo 40 cc/ mo Quinacrine; dihydroxyquinoline F Watery 2 years 40 cc/mo Tetracycline 3 24 F Intermittent 4 years 40 cc/mo Tetracycline Nodular Iymph F None" None Antiemetics oid hyper M Loose stools 6 years None None plasia Hypogamma M Intermittent 6 years 40 cc/mo Corticosteroids globulinemic 7 46 M Watery 12 years None Gluten-free sprue diet; corticosteroids for 10 years 8 e 37 F Watery 10 years 40 cc/mo Gluten-free diet a Normal values in milligrams per 100 ml: IgG, 770 to 1130; IgM, 90 to 170; IgA, 80 to 200, " Vomiting was this patient's main symptom; she had steatorrhea but no diarrhea. e Not infected with Giardia lamblia. mg/100 m(

3 218 AMENT AND RUBIN Vol. 62, No.2 length of the small intestine was sampled. Before each biopsy the tube's position was determined by fluoroscopy. In each case, one of the multiple biopsies was studied for Giardia Lamblia prior to fixation. It was placed with its cut surface against the index finger and the luminal surface was then wiped successively with two glass slides. The slides were then air dried for 1 hr, fixed in methanol for 30 min, washed in tap water, stained with Giemsa, and scanned for Giardia. Biopsies were carefully unfolded and oriented on a fine mesh of Saran plastic (available from National Filter Media Corp., Salt Lake City, U.). The biopsies were fixed in Bouin's solution, embedded in paraffin, and sectioned serially and completely. The stained slides from each biopsy were coded and interpreted blindly by the two authors. The biopsies were classified morphologically into four different groups: normal, mild abnormality, moderate abnormality, and severe abnormality. Fecal fat was determined on an aliquot of a 72-hr stool collection by the method of van de Kamer 23 ; the patients were eating 80 g of fat per day for 2 days prior to the collection and throughout the 3 days of the collection. A 21/2-year-old patient was fed a diet containing 50 rather than 80 g of fat per day. Fasting serum carotene levels were determined by the Price and Carr reaction. 2 ' Serum folate levels were determined by a microbiological assay with Lactobacillus casei (Dr. Robert Hillman's laboratory at the Harborview Medical Center performed these determinations.) In 2 cases before treatment and in 1 case afterwards, protein loss into the gastrointestinal tract was determined after intravenous injection of SlCrCl 3 by the method of Rootwelt. 25 Lactose tolerance tests were performed after feeding the patient 100 g of lactose in a 10% solution. True blood glucose was determined in venous blood fasting and at four 1/2 -hr intervals afterwards. A rise of greater than 20 mg per 100 ml over the fasting value was considered normal. Small intestinal biopsies for disaccharidase determinations were taken at the duodenojejunal junction, wrapped in parafilm, placed in small tightly capped plastic containers and frozen at -70 C. They were packed and shipped in dry ice for assay. (Dr. Gary Gray's laboratory at Stanford University performed the disaccharidase determinations.) Standard Schilling urinary excretion tests were performed without added intrinsic factor in 7 patients. 26 Bacterial cultures of proximal jejunal contents were obtained with a sterile sump tube to which a mercury bolus was attached. Following an overnight fast the tube was passed perorally into the stomach and small intestine. Before aspiration the position of the mercury bolus was checked fluoroscopically. A volume of fluid equal to the dead space of the tube was aspirated and discarded. Another 5-cc sterile syringe was used to aspirate a minimum of 1 cc. Mter the syringe was disconnected and capped with a sterile needle, all air bubbles were expressed from the aspirate. The time elapsed between procuring the specimen and placing it on all the media averaged 25 min and never exceeded 40 min. The specimens were inoculated on plates using the following media: calf serum-enriched trypticase soy yeast (TSY) broth [TSY-Baltimore Biological Laboratories (BBL) base-yeast extract]; TSY (BBL) agar deeps; blood agar plates (BBL);, J:ilood agar-neomycin plates (BBL) (neoi;ilycib 30 ILg per ml); Nagler plates (Bacto-peptQnirbase); MacConkey agar (without crysta;l\,;{oiet, final concentration of Neutral Red ;,.~ : 7; ILg per ml); and Sabouraud's agar (Sabot1hlUd's dextrose agar, BBL-Emmon's modification with addition of chloramphenicol, 4 ILg per ml). All media were incubated anaerobically at 35 C in a gas mixture of 85% H 2, 5% N 2, and 10% CO 2 for 4 days in Torbal jar containing a cold-catalyst. One cubic centimeter of specimen was mixed by inversion with TSY-serum-enriched broth and serially diluted to 10 9 ; of these dilutions cc (by calibrated loop) was cultured on MacConkey whole plates. Aliquots (0.001 cc) of undiluted and diluted (10-3) specimens were cultured on neomycin blood agar and plain blood agar plates; they were incubated anaerobically. Undiluted specimen (1/30 cc) was cultured on BAIMAC bi-plate and incubated aerobically and anerobically. Undiluted specimen [0.05 cc (from noncalibrated loop)] was cultured on Nagler's plates, onehalf of which had been prestreaked with Clostridium perfringens antitoxin. Organisms were identified by colonial morphology, growth on selective media, and reaction to Gram stain. Subculture and further identification were performed on microorganisms which were not readily classified by the above techniques. The presence of fungus was determined by Gram staining colonies present on Sabouraud's agar with chloramphenicol. The number of microorganisms per milliliter of intestinal juice was expressed as the logarithm to the base 10 of

4 February 1972 GIARDIASIS IN GASTROINTESTINAL IMMUNODEFICIENCY SYNDROMES 219 the actual number of organisms found. In certain bacteria it was impossible to calculate an accurate absolute colony count because of overgrowth; in these instances relative quantitation of 1 to 4+ was used. A control group of ten healthy males and females without antibody deficiency syndromes or gastrointestinal symptoms.was cultured in the same manner as the cases studied. Serum immunoglobulins were quantitated (Dr. Starkey Davis' laboratory at the University of Washington performed these determinations) by radial diffusion using commercially available plates (Melpar Biological Products Laboratory, Falls Church, Va.) containing IgG, IgA, and IgM.27 Results were compared to standard curves and to simultaneously run pooled normal serum. After an overnight fast gastric analyses were performed in all patients. A sump tube with attached mercury bolus was passed into the gastric antrum under fluoroscopic control. The gastric contents were aspirated and tested for acid with Topfer's reagent. Any patient who did not have acid in gastric contents was given a Histalog (Histalog (50 mg per cc), Eli Lilly and Co.) stimulation test (1.5 mg per kg) and continuous gastric aspiration was performed for 90 min afterward. Seven patients received a 6- to 8-week course of metronidazole, 250 mg three times a day. One of these 7 patients had a flat lesion and severe malabsorption but no giardiasis (patient 8); she was treated as a control to test the possible specific beneficial effect of metronidazole on the intestinal lesion. Because of her small size patient 4 was treated with 375 mg of metronidazole daily but this treatment had to be abandoned before Giardia Lamblia were eradicated because of side effects of fever and arthritis. Therefore this patient was retreated with quinacrine, 1.0 g per day for 10 days. Results Giardia infestation was present in all patients with mixed lesions and nodular lymphoid hyperplasia; 2 of the 3 patients with hypogammaglobulinemic sprue also were infected. In our 7 patients with giardiasis the diagnosis was made correctly by small intestinal biopsy and biopsy smear but only 4 of these same 7 patients could be diagnosed by stool examination (table 2). Prior therapy (table 1) did not eradicate giardiasis in our 7 patients and did not improve their condition substantially. On the other hand, the clinical improvement after eradication of Giardia lamblia was often dramatic (table 3): patient 7 had intractable severe diarrhea and steatorrhea for 10 years that became life-threatening. Only large doses of corticosteroids controlled his malabsorption sufficiently to keep him alive. After eradication of his giardiasis he lost his steatorrhea and gained 12 kg, despite discontinuance of corticosteroids. Five of our patients with giardiasis had diarrhea and all improved when their giardiasis was eradicated. The remaining patient (patient 4) with recurrent vomiting and steatorrhea lost her symptoms after her giardiasis was successfully treated. In the 4 patients with steatorrhea and giardiasis (patients 1, 2, 4, 7), normal fat absorption was restored by successful treatment of the infestation and all had significant weight gain (table 3). Serum carotene increased and serum folate became normal in 4 patients. The 1 patient without giardiasis who had steatorrhea (patient 8) failed to gain weight or show improvement in any of the laboratory tests after prolonged treatment with metronidazole. Vitamin B12 absorption as judged by the urinary excretion test of Schilling was borderline or abnormal in 6 patients with giardiasis (table 3). There were two clearly abnormal vitamin B12 absorption TABLE 2. Comparison of three methods used to diagnose giardiasis Stools examined Smears of Group Patient Biop biopsy sieso ' mucuso No. No. positive Mixed lesions Nodular Iymph oid hyper plasia 5 Hypogamma Not done globulinemic sprue 8" a +, positive; -, negative. " Only patient without giardiasis.

5 220 AMENT AND RUBIN Vol. 62, No.2 TABLE 3. Laboratory tests before and after treatment of giardiasis Fecal fat Weight (normal, Group lpatien < 6.0 g/day) Before Mter Before After kg g/jay Mixed lesions Nodular lymphoid hyperplasia Carotene Folate Protein loss BI2 Urinary (normal, (normal. (normal, excretion "g >5 m"g/ml) <50 ml/day) (normal, per 100 ml) >15%) Before Mter Before After Before Mter Before After,.g/100 ml m"g/ml ml/day % < 1.0 < Hypogammaglobulinemie sprue a a Only patient without giardiasis. tests (patients 2 and 7) and only 1 improved after treatment. Treatment with metronidazole clearly improved vitamin B 12 absorption in only 2 of the remaining 4 patients. The lactose tolerance test was abnormal before treatment in 4 out of 6 patients tested; 3 of the 4 had giardiasis. Diarrhea developed in 3 of the 4 patients during the test. Lactose tolerance returned to normal in the 3 patients whose giardiasis had been eradicated by treatment but remained abnormal in the 1 patient who was not infected (table 4). Before treatment disacc'baridases were reduced in 3 of the 5 patients tested. Two of these 3 patients had giardiasis. Disaccharidases returned to normal in the biopsies of those 2 patients where Giardia had been eradicated (table 5). Three hundred eighty four intestinal biopsies were taken during this study. Prior to treatment there was a wide spectrum of abnormalities of villus architecture in all of our patients. The severity of abnormalities varied greatly within each patient; i.e., the lesions were patchy (table 6). After treatment all of the biopsies in patients with mixed lesions had improved histologically. Villus architecture improved in 1 of our patients with nodular lymphoid hyperplasia and was not very abnormal to begin with in the other; lymphoid nodules persisted despite treatment. Two patients with giardiasis and the flat lesion of hypogammaglobulinemic sprue exhibited dramatic improvement after treatment. The 3rd patient with this entity did not have giardiasis and exhibited no improvement whatsoever after a long course of metronidazole. TABLE 4. Lactose tolerance tests before and after treatment of giardiasis Maximum rise. of glucose (normal, Group Patient >20 mg per 100 ml) Before mg/loo ml After Mixed lesions Nodular lymphoid hyperplasia Hypogammaglobulin- 7 7 ernie sprue 8 a 3-5 a Only patient without giardiasis.

6 February 1972 GIARDIASIS IN GASTROINTESTINAL IMMUNODEFICIENCY SYNDROMES 221 Before treatment the predominant mixed lesion in the proximal jejunum of patient 1 was of moderate severity (fig. la). After treatment most of his biopsies were normal (fig. IB). Before treatment in patient 2 the mixed lesion in the proximal jejunum was mostly moderate or severe and one of his moderate lesions is illustrated (fig. 2A); after treatment his lesions were mostly mild (fig. 2B). Before treatment in patient 7 with hypogammaglobulinemic sprue the lesion in the proximal jejunum was severe (fig. 3A) as shown in numerous biopsies taken over 10 years before his giardiasis was diagnosed and had been documented; after treatment his intestinal pathology regressed remarkably to a mild lesion (fig. 3B). In patient 1 with a mixed lesion, biopsies were also taken along the full length of the small intestine. The distal ileum was normal both before and after treatment and the distal small intestine in general had less severe lesions than the proximal intestine. Nevertheless the distal small intestine also improved after treatment. Before treatment patient 7 with hypogammaglobulinemic sprue had the most severe malabsorption in this series and the whole length of his small bowel exhibited a "severe flat" lesion. Cultures of intestinal fluid aspirated near the duodenojejunal junction were abnormal in 5 of the 8 patients studied. On the three occasions where it was possible to study the patients before and af- TABLE 5. Intestinal disaccharidases before and after treatment of giardiasis Lactase Sucrase Maltase Sucrase to lactase (normal. ;, 1 (normal, ;,3.5) (normal, ;, 10) ratio (normal Group Patient 0.9 to 4.5) Before After Before After Before After Before After U/ g wet weight U/g wet weight U/g.wet weight Mixed lesions S.l S ls Nodular lymphoid hyper plasia Hypogammaglobulinemic sa sprue a Only patient without giardiasis. TABLE 6. Proximal jejunal intestinal biopsies before and after treatment of giardiasis Group Patient Biopsy Normal Mild Moderate Severe Mixed lesions 1 Before 3 S 15 S Mter Before After Before After Nodular lymphoid hyperplasia 4 Before Mter Before After Hypogammaglobulinemic sprue 6 Before Mter Before After sa Before After a Only patient Without giardiasis.

7 222 AMENT AND RUBIN Vol. 62, No.2 FIG. 1. A, representative biopsy near the duodenojejunal junction taken from patient 1 before eradication of giardiasis. The abnormality of villus architecture is of moderate severity. B, representative biopsy after eradication of giardiasis. Taken from same area in patient 1. Villus architecture is normal (x 120). ter metronidazole treatment there was no change in intestinal flora except that Bacteroides g'ppeared in 1 patient after his giardiasis was treated (table 7). Six of the 8 patients had acid in their fasting gastric aspirates. The other two had Histalog-fast achlorhydria. Discussion Seven of our 8 patients with gastrointestinal immunodeficiency were infected with Giardia Lamblia. Plasma cells were virtually absent in the small intestinal biopsies of all these patients. The villus architecture in multiple small intestinal biopsies from the same patient often varied from normal to severely abnormal. Because of this patchy abnormality, mucosal response to eradication of Giardia Lamblia required many biopsies, both before and after treatment. Probably much of the past confusion regarding the relationship between giardiasis and abnormalities in intestinal structure is explained by the patchy nature of the lesion which could be easily missed in a single biopsy. We had always thought that patients with so-called hypogammaglobulinemic sprue had a severe intestinal lesion with severe malabsorption which was particularly resistant to. treatment. What we had not appreciated was the insensitivity of stool examination in diagnosing giardiasis. Once we discovered trophozoites of Giardia Lamblia in the sections and

8 February 1972 GIARDIASIS IN GASTROINTESTINAL IMMUNODEFICIENCY SYNDROMES 223 smears of intestinal biopsies in patient 7, we had something to treat, and the., histological and clinical improvement was remarkable. We now realize that many, but not all, of the intestinal abnormalities in immunodeficiency syndromes are related to giardiasis. Previous histological classifications may therefore be invalid except for nodular lymphoid hyperplasia because lymphoid nodules are probably unaffected by eradicating the Giardia. One of our patients who was free of giardiasis nevertheless had severe abnormalities in intestinal structure and function. This patient's abnormalities did not respond to a prolonged course of metronidazole, suggesting that the improvement in our other 7 patients was not an effect of the drug per se. Furthermore the presence of a severe intestinal lesion despite the absence of Giardia Lamblia indicates that other pathogenetic mechanisms must also be operative. Indeed, the intestinal structural abnormalities did not disappear completely in any of our patients although they did improve after eradication of Giardia lamblia. One wonders whether proximal intestinal bacterial overgrowth might not have been another factor contributing to the pathogenesis of the intestinal lesion as it does in stasis syndrome. 28 In support of this speculation was o~r observation of the most dramatic reversion towards normal FIG. 2. A, representative biopsy near the duodenojejunal junction taken from patient 2 before eradication of giardiasis. The abnormality of villus architecture is of moderate severity. B, representative biopsy after eradication of giardiasis. Taken from same area in patient 2. Villus architecture shows a mild abnormality (x 120).

9 224 AMENT AND RUBIN Vol. 62, No.2 FIG. 3. A, biopsy taken near the duodenojejunal junction from patient 3, which was representative of the severe abnoi;mality in villus architecture observed repeatedly in this patient over a period of 10 years prior to the diagllosis of giardiasis. B, representative biopsy after eradication of giardiasis. Taken from same area in patient 3. Villus architecture shows a mild abnormality (x 120). after eradicating Giardia Lamblia in 1 patient without bacterial overgrowth (patient 1). The improvement in absorptive capacity after eradication of Giardia lamblia paralled improvement in intestinal structure. When present, steatorrhea disappeared and absorption of carotene and folate apparently improved. This suggests that much of the malabsorption might be explained by intestinal mucosal injury. The malabsorption of vitamin B12 was far more complex. In patient 7 ileal injury probably explained the malabsorption before treatment. However vitamin B12 absorption improved in only 2 of the remaining 5 patients after Giardia lamblia were eradicated. Perhaps overgrowing bacteria were binding intrinsic factor or utilizing vitamin B 1 2 One of the least understood areas of malabsorption is abnormal growth of bacteria or parasites within the lumen with little apparent invasion of the intestine

10 February 1972 GIARDIASIS IN GASTROINTESTINAL IMMUNODEFICIENCY SYNDROMES 225 TABLE 7. Small intestirwl bacterial flora at the duodenojejurwl junction before and after treatment of giardiasis Group Clostrid Escherichia colia Bacteroides specieso ium per- Enterococcia Pa (normal, O) (normal,o) fringensa (normal,o) (normal, O) tient Before After Before After Before After Before After Mixed lesions ND" 3.0 x 10 7 ND 0 ND 2+ ND 1.0 x 10" 3 ND 2.7 x 10" ND 2+ ND 1+ ND Benign nodular X hyperplasia Hypogammaglobu linemic sprue 7 ND 0 ND 3+ ND 2+ ND 2+ 8 e 2.2 X 10" 4.7 x 10" 1.0 X 10' x 10' 2.1 X 10' a No colonies were isolated from 10 normal controls. " ND, not done. e Only patient without giardiasis. itself. Examples of this type of malabsorption are giardiasis, capillariasis 29 and stasis syndrome. Intestinal structure may be altered in all three of these entities. Improvement in intestinal structure by light microscopy after eradication of the organisms has only been shown in giardiasis in the presence of immune defects. Nevertheless intestinal function improves in all three entities. Much more work remains to be done before there is complete understanding of the basic mechanisms of malabsorption in any of the various types of abnormal luminal overgrowth. REFERENCES 1. Twomey JJ, Jordan PH, Jarrold T, et al: The syndrome of immunoglobulin deficiency and pernicious anemia. Am J Med 47: , Sanford JP, Favour CB, Tribeman MS: Absence of serum gamma globulin in adult. N Engl J Med 250: , Rosecan M, Troubaugh FE Jr, Danforth WH: Agammaglobulinemia in adult. Am J Med 19: , Waldman TA, Schwab PJ: IgG (75 gamma globulin) metabolism in hypogammaglobulinemia: studies in patients with defective gamma globulin synthesis, gastrointestinal protein loss, or both. J Clin Invest 44: , Allen GE, Hadden DR: Congenital hypogammaglobulinemia with steatorrhea in two adult brothers. Br Med J 2:486:490, Cohen N, Paley D, Janowitz, HD: Acquired hypogammaglobulinemia and sprue: Report of a case and review of the literature. J Mount Sinai Hosp 28: , Crabbe PA, Heremans JF: Lack of gamma-aimmunoglobulin in serum of patients with steatorrhea. Gut 7: , Forssman 0, Herner B: Acquired agammaglobulinemia and malabsorption. Acta Med Scand 176: , Hoskins LC, Winawer SJ, Broitman SA, et al: Clinical giardiasis and intestinal malabsorption. Gastroenterology 53: , Huizenga KA, Wollaeger EE, Green PA, et al: Serum globulin deficiencies in nontropical sprue with report of two cases of acquired agammaglobulinemia. Am J Med 31: , 1961 II. Pelkonen R, Siurala M, Vuopio P : Inherited agammaglobulinemia with malabsorption and marked alterations in the gastrointestinal mucosa. Acta Med Scand 173: , Collins JR, Ellis DS: Agammaglobulinemia, malabsorption and rheumatoid-like arthritis. Am J Med 39: , Hermans PE, Huizenga KA, Hoffman HN, et al: Dysgammaglobulinemia associated with nodular lymphoid hyperplasia of the small intestine. Am J Med 40:78-89, Kirkpatrick GH, et al: Hypogammaglobulinemia with nodular lymphoid hyperplasia of the small bowel. Ann Intern Med 121: , Rosen FS, Janeway CA: The gammaglobulins. II. The antibody deficiency syndromes. N Engl J Med 275: , Rubin CE, Eidelman S, Weinstein WM: Sprue by any other name. Gastroenterology 58: , Alp MH, Hislop IG: The effects of Giardia lam-

11 226 AMENT AND RUBIN Vol. 62, No.2 blia infestation on the gastrointestinal tract. Australas Ann Med 17: , Yardley JH, Takano J, Hendrix TR: Epithelial and other mucosal lesions of the Jejunum in giardiasis jejunal biopsy studies. Bull Johns Hopkins Hosp 115: , Fudenburg HH, Good RA, Hitzig W, et al: Classification of the primary immunodeficiencies: W. H. 0. recommendation. N Engl J Med 283: , Faust EC, Russell PF: Clinical Parasitology. Seventh edition. Philadelphia, Lea and Febiger, 1964, p 91-94, Brandborg LL, Rubin CE, Quinton WE: A multipurpose instrument for suction biopsy of the esophagus, stomach, small bowel, and colon. Gastroenterology 37: 1-16, Flick AL, Quinton WE, Rubin CE: A peroral hydraulic biopsy tube for multiple sampling at any level of the gastrointestinal tract. Gastroenterology 40: , Van der Kamer JH, ten Bokkel Huinink H, Weijers HA: Rapid method for the determination of fat in feces. J Bioi Chem 177: , Varley H: Practical Clinical Biochemistry. Third edition. London, Heinemann, 1962, p Rootwelt K: Direct intravenous injection of "chronic chloride compared with 125I_poly_ vinylpyrrolidone and 13II-albumin in the detection of gastrointestinal protein loss. Scand J Clin Lab Invest 18: , Schilling RF: Intrinsic factor studies. II. The effect of gastric juice on the urinary excretion of radioactivity after the oral administration of radioactive vitamin B 12 J Lab Clin Med 42: , Mancini G, Carbonara AO, Heremans JF: A single radial diffusion method for immunological quantitation of proteins. Prot Bioi Fluids 11:370, Ament ME, Shimoda SS, Saunders DR, et al.: The pathogenesis of steatorrhea in 'stasis syndrome (abstr). Gastroenterology 60:637, Whalen GE, Rosenberg EB, Strickland GT, et al: Intestinal capillariasis: a new disease in man. Lancet 1:13-16, 1969