IMMUNOLOGICAL STUDIES IN ACUTE PSEUDOMEMBRANOUS ESOPHAGEAL CANDIDIASIS

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1 0016~5085/78/ $02.00/0 GABTROENTEROMGY 75: , 1978 Copyright by the American Gastroenterological Association Vol. 75, No. 2 Printed in USA. CASE REPORTS IMMUNOLOGICAL STUDIES IN ACUTE PSEUDOMEMBRANOUS ESOPHAGEAL CANDIDIASIS SUDHIR K. DUTTA, M.D., AND MOHAMED S. AL-IBRAHIM, M.D. Divisions of Gastroenterology and Infectious Diseases, University of Maryland School of Medicine, and Veterans Administration Hospital, Baltimore, Maryland Immunological studies in 3 patients with acute pseudomembranous esophageal candidiasis are presented. of the patients had history or clinical signs of chronic mucocutaneous candidiasis. All 3 patients had an intact humoral response as measured by the presence of elevated candida agglutinating antibody titers. However, further investigations revealed a cell-mediated immune defect characterized by in vivo and in vitro anergy to candida antigen in these patients. Response to other antigens was found to be intact in 2 of 3 patients. Successful medical therapy was associated with return of skin and lymphocyte reactivity to candida antigen in two cases. Esophageal candidiasis is an increasingly recognized clinical entity. l, 2 Most cases are found to have underlying malignancy,3 and endocrine or immunological disorders,4* 5 whereas a minority are associated with the prolonged use of broad spectrum antibiotics.6 Corticosteroids and other immunosuppressives also predispose to this condition.7 The esophageal involvement is frequently acute, but may also be chronic as a feature of chronic mucocutaneous candid&is (CMC). Occasionally, it may be associated with a life-threatening disseminated candida infection.4, 8 We recently encountered 3 patients who presented with esophageal candidiasis (acute pseudomembranous type) without any evidence of skin, nail or hair involvement. These patients were investigated for (1) a possible impairment of cellular and/or humoral immunity, and (2) the effect of antifungal therapy on the immunological status. The results of these studies constitute the basis of this report. Patients and Methods All 3 patients were white males who presented with a history of dysphagia and/or odynophagia of varying duration. The diagnosis of acute pseudomembranous esophageal candidiasis was made by esophagoscopy with demonstration of fungal mycelia on direct smear and/or mucosal biopsy, positive candida cultures of esophageal brushings, and high candida agglutinin titers. Each patient was studied immunologically Received September 15, Accepted January 25, Address requests for reprints to: Sudhir K. Dutta, M.D., Veterans Administration Hospital, 3900 Loch Raven Boulevard, Baltimore, Maryland This study was supported in part by the Medical Research Service of the Veterans Administration, and by a grant from the Pangborn Fund. The authors wish to thank Mrs. Victoria Currid for excellent technical assistance and Mrs. Helen Spencer for preparation of the manuscript. 292 before, immediately after, and 3 months after successful therapy. If there was recurrence of the disease, then the diagnostic and immunological evaluation were repeated in an identical manner. All immunological studies were performed 3 to 5 days after disappearance of ketosis and stabilization of blood sugar levels. The clinical information on each case is summarized in table 1. Immunological Studies Mononuclear cells (lymphocytes and monocytes) were obtained from heparinized peripheral blood after separation on Fic.oll-Hypaque according to the method of Boyum.s Lymphocyte transformation. Cells were adjusted to 0.75 x 10fi lymphocytes per ml in RPM (Microbiological Associates, Bethesda, Md.). The final culture medium contained penicillin and streptomycin (100 U and 100 pg per ml, respectively) and 15% autologous plasma. The cells were cultured in microtiter plates (Linbro Chemical Co., Inc., New Haven, Corm.), each flat-bottomed well contained 0.2 ml of the cell suspension. Tuberculin (PPD, Connaught Medical Research Laboratories, Willowdale, Ontario, Canada) was added to final concentrations of 1, 2.5, 5, and 10 pg per ml. Preservative-free candida antigen (CAg, Hollister-Stier Laboratories, Downers Grove, Ill.) was used in the following manner: 10, 20, and 30 ~1 of stock solution per well; dilutions of 1:2 and 1:4 were also tested. Streptokinase-streptodornase (SK-SD, Varidase, Lederle Laboratories, Pearl River, N. Y.) was added to a final concentration of 40, 80, and 120 U per ml. In addition to the above, lymphocyte reactivity to the mitogens concanavalin A and pokeweed was also studied. All cultures were incubated at 37 C in an atmosphere of 5% CO,-air. During the final 18 hr of incubation with antigen 01: mitogen, [3H]thymidine was added to a final concentration of 2 &i per ml. The cells were transferred to filter paper by means of an automated harvester, washed with distilled water, dried, and counted in a liquid scintillation counter. E-rosettes. These tests were performed according to the methods of Jondal et al. and Wybran et ali2 Lymphokine production. The indirect agarose leukocyte migration inhibition technique of Clausen13 was used to test for the presence of leukocyte inhibitory factor (LIF) in antigen-

2 August 1978 CASE REPORTS 293 TABLE 1. Summary of clinical features, diagnostic tests, therapy, and follow-up in 3 patients with acute pseudomembranous esophageal candidiasis Age (yr) Duration of diabetes mellitis (DM) (yr) Daily msulin requirement Recurrent infections Ketacidosis Retinopathy Neuropathy Control of blood sugar U NPH 35 U Lente 52 U NPH One episode before present illness Good Presenting complaint Odynophagia for 2 wk Oral thrush Nail, skin, or hair infection Hematocrit Barium swallow Esophagoscopy Esophageal brushing cul- tures Esophageal biopsy Theraputic response Oral nystatin Amphotericin B Follow-up cl Neutral protamine Hagedorn. Patients R. S N. R Normal Normal Esophagitis with yellowish plaques Esophagitis with yellowish plaques overlying inflamed mucosa in overlying inflamed mucosa in distal half of esophagus distal half of esophagus Candida albicans Esophagitis with fungal invasion Severe esophagitis Not used lo-mo duration, asymptomatic; DM well controlled Recurrent episodes during active candida esophagitis, none since the control of esophagitis Grade II Mild Difficult only during candida esophagitis Mild hematemesis and ketoaci- dosis Seen on one admission Candida tropicalis 8 mo, asymptomatic; no episodes of hematemesis and ketoacidosis; DM under control B. C. Aspiration pneumonia twice; urinary bladder infection twice sec- ondary to catheterization nary incontinence 35 Distal esophageal stricture Esophagitis with yellowish plaques overlying the inflamed mucosa in distal half of esophagus along with marks distal esophageal narrowing Candida albicans Severe esophagitis vasion for uri- Grade II Severe (autonomic nervous system involvement) Difficult during period of infection Dysphagia to solid foods for 4 wk with fungal in- 6 mo, asymptomatic; esophageal stricture dilated, DM well under control; no episode of pneumonia or urinary tract infection treated lymphocyte cultures. Supernatants were prepared by incubating 4 x 10 lymphocytes in 2 ml of RPM with 10% horse serum for 72 hr with or without antigen. The areas of migration were calculated for each well and the mean of four migration areas was obtained for each sample. Migration was expressed as the LIF index and was calculated as follows: mean area of migration in supernatants of antigen-treated cultures LIF index = - mean area of migration in control supernatants x100 In our laboratory, significant leukocyte migration inhibition correlates with LIF index of 80 or less. B lymphocytes. Labeling of immunoglobulin-bearing lymphocytes was performed according to the method of Jondal et al. Delayed hypersensitivity. Skin testing was performed by 0. l-ml intradermal injection of intermediate PPD (5 tubercu- lin U), candida antigen l:lo,ooo and l:l,ooo, and 40 U of SK- SD. All skin reactions were evaluated 48 hr after injection and only induration was measured. Serum candida agglutinin titers were kindly performed by Dr. Kozinn. 1a Results No past history of candida infection or other fungal diseases was elicited in any patient. Further, no signs of CMC or clinical evidence of parathyroid or adrenal gland hypofunction were detected in any case. The serum calcium, phosphorus, and alkaline phosphatase were normal in all patients. Because of symptoms of postural hypotension, patient N. R. underwent extensive adrenal function evaluation which revealed normal adrenal cortical function. The control of blood sugar

3 294 CASE REPORTS Vol. 75, No. 2 levels was not difficult in our patients after the successful treatment of esophageal candiasis. The information on other complications of diabetes mellitus (DM) in these patients in provided in Table I. Immunological Studies Serum protein and immunoelectrophoresis profiles were normal for all 3 patients. Absolute lymphocyte and monocyte counts were normal and the number of T and B lymphocytes were within the range of our laboratory normal controls. Table 2 summarizes the results of the skin tests and candida antibody titers. All patients had significant candida agglutinating antibody titers at initial evaluation. It should be noted that R. S. and N. R. had intact delayed cutaneous hypersensitivity to PPD and SK-SD at the outset, but reactivity to CAg was absent. R. S. developed a positive skin reaction to CAg after nystatin therapy; in the case of N. R., however, a positive candida skin reaction became ap- TABLE 2. Results of various skin tests and candida antibody tiers in acute pseudomembranous esophageal candid&is before and after therap_y I Patient Skin tests (noninduration) Candida antibody titers CAg PPD SK-SD Agglutinin Precipitin R. S. NR 17 ND 1:320 Negative R. S. 11 ND 12 1:160 Negative R. S. 10 ND 18 ND ND N. R. NR 22 8 ND ND N. R.O NR ND 10 1:1280 Negative N. R. 8 ND 15 1:320 Negative B. C. NR NR NR 1:320 + B.C. NR NR NR 1:320 ND B. C. NR ND <4 ND B. C. NR ND 6 ND ;D fl Abbreviations are: CAg, candida antigen; PPD, tuberculin; SK- SD, streptokinase-streptodornase; NR, no reaction; ND, not done. 0 Post nystatin treatment. c Post amphotericin B treatment. parent only after amphotericin B therapy. Reactivity to CAg in B. C. remained negative even after systemic antifungal therapy. Interestingly, B. C. had positive candida precipitin antibody titer which is often associated with deep or systemic candidiasis. As regards lymphocyte function tests, it can be seen in table 3 that initially candida antigen did not induce a significant lymphocyte proliferation response in any of the patients. This was true for all concentrations of CAg tested. In contrast, the lymphocytes of cases 1 and 2 responded to PPD. In case 3, however, no significant lymphocyte reactivity to any of these antigens was seen. As can be noted, lymphokine production in response to antigen stimulation of lymphocytes (as measured by inhibition to leukocyte migration) paralleled lymphocyte proliferation and skin reactivity. Lymphocyte stimulation and LIF indices for CAg changed dramatically with clinical recovery after treatment in cases 1 and 2 and were accompanied by conversion of skin reactivity to that antigen. Case 3 continued to be nonreactive even after apparently successful antifungal therapy. In all 3 patients, lymphocyte proliferation in response to mitogen was normal. In order to test for the presence of plasma inhibitors, the patients cells were thoroughly washed and cultured in the presence of 15% pooled normal human serum. No differences in reactivity were noted between these cells and those incubated in autologous serum or plasma. Discussion Among the various infections involving the esophagus, candidiasis is by far the most common.16 Excluding those cases associated with the use of broad spectrum antibiotics, esophageal candidiasis generally indicates some defect of host immunity. Although a few cases have been described without an apparent predisposing factor, the detailed immunological investigations were not carried out in these patients.2, I7 Available data have revealed the presence of candida antibodies in esophageal infection with this organism, suggesting an appropriate humoral response.7 High TABLE 3. Lymphocyte reactivity in acute pseudomembranous esophageal candidiasis before and after therapy Patient Unstimulated cells C& PPD SK-SD countslmin R. S * ( ) 67 ( ) 8 (ND) R. S (73 f 9) 147 (ND) 34 (ND) R. S (68? 11) 135 (ND) 66 (ND) N. R (111 % 18) 140 ( ) 26 (75 11) N. R (103 * 12) 122 ( ) 37 (72? 16) N. R ( ) 261 (ND) 76 (ND) B. C ( ) 2 ( ) 32 (92 * 10) B. C (113 t 16) 7 (101? 8) 28 (93 f 11) B. C (ND) 1.2 (ND) 31 (ND) B. C (104 Y? 11) 0.8 (ND) 8 (ND) I Abbreviations are: CAg, candida antigen; PPD, tuberculin; SK-SD, streptokinase-streptdornase; ND, not done. h Stimulation index for lymphocytes. c Leucocyte inhibitory factor index (~~_sem). '! Post nystatin treatment. + Post amphotericin B treatment.

4 August 1978 CASE REPORTS 295 candida agglutinin titers were found in all 3 patients before treatment. Cell-mediated immunity is assumed to be intact in acute pseudomembranous candida infection of the pharynx (thrush), but has not been comprehensively studied in acute infection with esophageal involvement. H Our patients with esophageal candidiasis had a clearly demonstrable defect in cell-mediated immune function. Initially, there was failure to respond to candida antigen in vivo and in vitro in 2 patients, who manifested appropriate responses to other antigens. Ability to react to CAg was noted only after successful therapy. In the case of N. R., this was achieved only after systemic antifungal treatment. It is noteworthy that CAg induced lymphocyte DNA synthesis and lymphokine production paralleled delayed skin reactivity in all patients studied. Patient B. C. has remained nonreactive in spite of apparent clinical response to amphotericin B therapy. Studies in patients with CMC have revealed a specturm of immunodeficiencies with or without the concomitant presence of plasma inhibitors. ls, 2o Follow-up in some patients has revealed complete or partial immunological reconstitution with chemo- and/or immunotherapy.x Our patients manifested cell-mediated immune defects similar to those described in CMC. Moreover, longitudinal studies in 2 patients have shown reversibility of these defects with successful therapy; this phenomenon has also been described in CMC.14* 2o It is possible that our 3rd patient, who remains anergic to CAg, has a more profound immunological defect, and the presence of esophageal stricture formation may indicate a persistent or deep-seated infection. Whether the initial specific cellular anergy to CAg represented a primary state of susceptibility to candida infection or were of a secondary nature related to antigenic load is not known at this time. The possibility of macrophage dysfunction in candida infections has been raised by some workers,21 but seems unlikely in patients R. S. and N. R., inasmuch as they exhibited normal reactivity to other antigens. The role of DM in our patients is not clear. It is generally accepted that diabetics may under certain circumstances show a greater frequency of fungal infections. Several investigators have reported immunological defects in association with DM. Marble and associatesz2 noted impaired phagocytic ability in patients with diabetic ketoacidosis, and more recently, Van Ossz3 reported the depression of phagocytosis in hyperglycemic media. Conversely, others have reported normal phagocytic ability but decreased chemotactic function in DM. In children with juvenile DM, no defect of phagocytosis of candida could be demonstrated in vitro.24 Moreover, there is little evidence to indicate any specific pattern of alteration in cell-mediated immunity in patients with DM. Whether conditions such as hyperglycemia, acidosis, or genetic factors have played a role in the over-all pathogenesis of esophageal infection in these patients needs further clarification. In our patients, however, the diabetes was well under control at the time of each immunological investigation. These findings suggest that patients with acute candida esophagitis may have defects in cell-mediated immunity specific for candida in spite of an apparently intact response to other antigens. Whether characterization of this defect in this group of patients may have a role in delineating the pathogenesis of esophageal candidiasis or play a part in the management of this clinical entity can only be established by longitudinal studies of larger numbers of patients. REFERENCES 1. Eras P. Goldstein MJ, Sherlock P: Candida infection of the gastrointestinal tract. Medicine (Baltimore), 51: , Kodsi BE, Wickremsinghe PC, Kozinn PJ, et al: Candida esophagitis: a prospective study of 27 cases. 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5 296 CASE REPORTS Vol. 75, No. 2 nity: reversal after amphotericin B therapy. Clin Exp Immunol , Valdimarsson H, Higgs JM, Wells RS, et al: Immune abnormalities associated with chronic mucocutaneous candidiasis. Cell Immunol6: , Twomey JJ, Waddell CC, Krantz S, et al: Chronic mucocutaneous candidiasis with macrophage dysfunction, a plasma inhibitor, and coexistent aplastic anemia. J Lab Clin Med 85: , Marble A, White HJ, Fernald AT: The nature of lowered resistance to infection in diabetes mellitus. J Clin Invest 17:423, Van Oss CJ: Influence of glucose levels on the in uitro phagocy- tosis of bacteria by human neutrophile. Infect Immun 4:54-61, Miller ME, Baker L: Leukocyte functions in juvenile diabetes mellitus: humoral and cellular aspects. J Pediatr 91: , 1972