Immunological studies in acute pseudomembranous esophageal candidiasis

0016~5085/78/7502-0292$02.00/0 GABTROENTEROMGY 75:292-296, 1978 Copyright 0 1978 by the American Gastroenterological Association

CASE

Vol. 75,No. 2

Printed in USA.

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, Veterans Administration

Hospital, Baltimore,

University of Maryland Maryland

School of Medicine,

and

Immunological studies in 3 patients with acute pseudomembranous esophageal candidiasis are presented. None 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, 1977. Accepted January 25, 1978. Address requests for reprints to: Sudhir K. Dutta, M.D., Veterans Administration Hospital, 3900 Loch Raven Boulevard, Baltimore, Maryland 21218. 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.

before, immediately after, and 3 months after successful therapy. If there was recurrence of the disease, then the diagnosticand immunologicalevaluationwere repeatedin an identicalmanner. All immunologicalstudieswere performed 3 to 5 days after disappearanceof ketosis and stabilization of

blood sugar levels. The clinical information summarized in table 1.

Immunological

on each case is

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 10filymphocytes per ml in RPM1 1640 (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 antigen292

August 1978

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CASE REPORTS

TABLE1. Summary of clinical features, diagnostic tests, therapy, and follow-up in 3 patients with acute pseudomembranous

esophageal

candidiasis Patients

B. C.

N. R

R. S 54 6

45 15

34 13

msulin Daily requirement Recurrent infections

35 U NPH”

35 U Lente

52 U NPH

None

None

Aspiration pneumonia twice; urinary bladder infection twice secondary to catheterization for urinary incontinence

Ketacidosis

One episode before present illness

Absent

Retinopathy Neuropathy

Absent None

Recurrent episodes during active candida esophagitis, none since the control of esophagitis Grade II Mild

Control of blood sugar

Good

Difficult

Age

(yr)

Duration of diabetes mellitis (DM) (yr)

only

during

candida

Grade II Severe (autonomic nervous system involvement) Difficult during period of infection

ketoaci-

Dysphagia

Oral thrush Nail, skin, or hair infection Hematocrit Barium swallow Esophagoscopy

Absent None

esophagitis Mild hematemesis and dosis Seen on one admission None

42 Normal Esophagitis with yellowish plaques overlying inflamed mucosa in distal half of esophagus

38 Normal Esophagitis with yellowish plaques overlying inflamed mucosa in distal half of esophagus

Esophageal tures Esophageal

Candida albicans

Candida tropicalis

Esophagitis

Severe esophagitis

Severe esophagitis vasion

None Excellent 8 mo, asymptomatic; no episodes of hematemesis and ketoacidosis; DM under control

None Excellent 6 mo, asymptomatic; esophageal stricture dilated, DM well under control; no episode of pneumonia or urinary tract infection

Presenting

Odynophagia

complaint

brushing biopsy

Theraputic response Oral nystatin Amphotericin B Follow-up

cl Neutral protamine

cul-

for 2 wk

with fungal invasion

Excellent Not used lo-mo duration, asymptomatic; DM well controlled

to solid foods for 4 wk

Absent None 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 with fungal in-

Hagedorn.

treated lymphocyte cultures. Supernatants were prepared by incubating 4 x 10” lymphocytes in 2 ml of RPM1 1640 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

lin U), candida antigen l:lO,OOO and l:l,OOO, and 40 U of SKSD. 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 x100

In our laboratory, significant leukocyte migration inhibition correlates with LIF index of 80 or less. B lymphocytes. Labeling of immunoglobulin-bearing lym-

phocytes 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-

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

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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 apTABLE 2. Results of various skin tests and candida antibody tiers in acute pseudomembranous esophageal candid&is before and after

therap_y‘I Skin tests” (noninduration)

Patient

Candida antibody titers

CAg

PPD

R. S. R. S.” R. S.”

NR 11 10

17 ND ND

ND 12 18

N. R. N. R.O N. R.”

NR NR 8

22 ND ND

8 10 15

B. C. B.C.” B. C.’ B. C.’

NR NR NR NR

NR NR ND ND

NR NR <4 6

SK-SD

Agglutinin

Precipitin

1:320 1:160 ND

Negative Negative ND

ND 1:1280 1:320

ND Negative Negative

1:320 1:320 ND ND

+ ND

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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

;D

fl Abbreviations are: CAg, candida antigen; PPD, tuberculin; SKSD, streptokinase-streptodornase; NR, no reaction; ND, not done. 0 Post nystatin treatment. c Post amphotericin B treatment. TABLE 3.Lymphocyte Patient

Among the various infections involving the esophagus, candidiasis is by far the most common.16Excluding 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

reactivity in acute pseudomembranous

Unstimulated cells

esophageal candidiasis

before and after therapy”

PPD

C&

SK-SD

countslmin 67 (74 + 14) 147 (ND) 135 (ND)

8 (ND) 34 (ND) 66 (ND)

1.2 (111 % 18) 1.0 (103 * 12) 175 (79 2 13)

140 (63 2 13) 122 (67 + 16) 261 (ND)

26 (75 ” 11) 37 (72 ? 16) 76 (ND)

147 3 69

1.0 (103 + 11) 2.8 (113 t 16) 3.8 (ND)

2 (97 2 14) 7 (101 ? 8) 1.2 (ND)

32 (92 * 10) 28 (93 f 11) 31 (ND)

85

1.1 (104 Y? 11)

0.8 (ND)

R. S. R. S.” R. S.”

182 158 139

N. R. N. R.” N. R.”

112 92 97

B. C. B. C.” B. C.’ B. C.’

0.7* (98 + 12)’ 47 (73 f 9) 39 (68 ? 11)

‘I Abbreviations are: CAg, candida antigen; PPD, tuberculin; h Stimulation index for lymphocytes. c Leucocyte inhibitory factor index (~~_sEM). '!Post nystatin treatment. +’Post amphotericin B treatment.

SK-SD, streptokinase-streptdornase;

8 (ND) ND, not done.

August 1978

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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), 51:367-379, 1972 2. Kodsi BE, Wickremsinghe PC, Kozinn PJ, et al: Candida esophagitis: a prospective study of 27 cases. Gastroenterology 71:715719, 1976 3. Jensen KB, Stenderup A, Thomsen JB, et al: Esophageal moniliasis in malignant neoplastic disease. Acta Med Stand 175:455459, 1964 4. Quie PG, Chilgren RA: Acute disseminated and chronic mucocutaneous candidiasis. Semin Hematol8:227-242, 1971 5. Hermans PE, Ulrich JA, Markowitch H: Chronic mucocutaneous candidiasis as a surface expression of deep-seated abnormalities. Report of a syndrome of superficial candidiasis, absence of delayed hypersensitivity and amino-aciduria. Am J Med 47:503-519, 1969 6. Seelig MS: Mechanisms by which antibiotics increase the incidence and severity of candidiasis and alter the immunological defenses. Bacterial Rev 30:442-459, 1966 7. Frenkel JK: Role of corticosteroids as predisposing factors in fungal diseases. Lab Invest 11:1192-1208, 1962 8. Kirkpatrick CH, Rich RR, Bennett JE: Chronic mucocutaneous candidiasis: model-building in cellular immunity. Ann Intern Med 74:955-978, 1971 9. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Stand J Clin Lab Invest 21:77-89, 1968 10 Al-Ibrahim MS, Holzman RS, Lawrence HS: Concentrations of levamisole required for enhanced proliferation of human lymphocytes and phagocymsis by macrophages. J Infect Dis 135:517523, 1977 11 Jondal M, Holm G, Wigzell H: Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells. J Exp Med 136:207-215, 1972 12. Wybran J, Carr MC, Fudenberg HH: The human rosette-forming cell as a marker of a population of thymus-derived cells. J Clin Invest 51:2537-2542, 1972 13. Clausen JE: The agarose migration inhibition technique for in vitro demonstration of cell-mediated immunity in man. A review. Dan Med Bull 22:181-194, 1975 14. Taschdjian CL, Seelig MS, Kozzin PJ, et al: Serological diagnosis of candidal infections. CRC Crit Rev Clin Lab Sci 4:19-59, 1973 15. Rosher F, Gabriel FD, Taschdjian CL, et al: Serologic diagnosis of systemic candidiasis in patients with acute leukemia Am J Med 51:54-62, 1971 16. Kramer P: Infections of the esophagus. In Gastroenterology, vol I. Edited by HL Bockus. Philadelphia, WB Saunders Co, 1974, p 330 17. Holt JM: Candida infection of the oesophagus. Gut 9:227-231, 1968 18. Feigin RD, Shearer WT: Opportunistic infection in children. I. In the compromised host. J Pediat 87:507-514, 1975 19. Paterson PY, Semo R, Blumenchein G, et al: Mucocutaneous candidiasis, anergy and a plasma inhibition of cellular immu-

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nity: reversal after amphotericin B therapy. Clin Exp Immunol 9595-602, 1971 20. Valdimarsson H, Higgs JM, Wells RS, et al: Immune abnormalities associated with chronic mucocutaneous candidiasis. Cell Immunol6:348-361, 1973 21. 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 85:968-977, 1975

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22. Marble A, White HJ, Fernald AT: The nature of lowered resistance to infection in diabetes mellitus. J Clin Invest 17:423, 1938 23. Van Oss CJ: Influence of glucose levels on the in uitro phagocytosis of bacteria by human neutrophile. Infect Immun 4:54-61, 1971 24. Miller ME, Baker L: Leukocyte functions in juvenile diabetes mellitus: humoral and cellular aspects. J Pediatr 91:979-982, 1972