Vol. 15, No. 1 Printed in U.S.A.
INFECTION AND IMMUNITY, Jan. 1977, p. 287-294 Copyright ©) 1977 American Society for Microbiology
Humoral Immunity in Vaginal Candidiasis1 SUBBI MATHUR,* G. VIRELLA, J. KOISTINEN, E. 0. HORGER III, T. A. MAHVI, AND H. HUGH FUDENBERG Department of Basic and Clinical Immunology and Microbiology,* and Department of Obstetrics and Gynecology, Medical University of South Carolina, Charleston, South Carolina 29401 Received for publication 9 September 1976
Serum antibody titers to Candida albicans were estimated in 37 women with recurrent vaginal candidiasis and in 148 normal American and Finnish subjects, using the passive-hemagglutination technique. The antibody titers ranged from 0 to 16 in normal individuals and 4 to 256 in vaginal candidiasis patients. Antibodies to C. albicans in the sera of vaginal candidiasis patients were found to be the secretary immunoglobulin A type, as determined by gel filtration and double-diffusion tests. The results were confirmed by the indirect fluorescentantibody technique. Our findings suggest that, in vaginal candidiasis, the antibody response is mainly local, consisting of secretary immunoglobulin A, some of which finds its way into systemic circulation.
Candida albicans, a saprophytic fungus belonging to the Cryptococcaceae, is a common constituent of the microflora in body cavities. However, it assumes the role of a severe pathogen under certain circumstances, such as malignancies (4, 16, 18, 19, 20), major surgery involving the use of immunosuppressants and broad-spectrum antibiotics (14), or pregnancy (5). In women, vaginal candidiasis is a common and chronic ailment. Since this fungus is a normal inhabitant of the vaginal area, microscope evidence alone may not suffice for detection of active infection. An effective serodiagnostic tool would therefore be useful and would allow some insight into the patient's humoral immune response to the fungus. Tube agglutination (1), immunodiffusion (6, 8, 18, 19, 21), counterimmunoelectrophoresis (2, 8), and indirect fluorescent-antibody techniques (6, 7, 13, 15, 21) have been used, largely in studies on systemic candidiasis. Precipitin techniques yield positive results in 89 to 95% of cases (8, 14, 21). Agglutination techniques have been demonstrated to be more sensitive, and positive precipitins by double diffusion usually correspond to agglutination titers of 1:60 to 1:256 (1, 18, 19). There is little information available, however, in regard to the humoral immunity of vaginal candidiasis patients. Double diffusion has been shown to be insufficiently sensitive (20), and previous studies with agglutination methods (5, 12, 24, 26) have not been
The present paper describes the characterization of circulating antibodies in patients with vaginal candidiasis and the application of the passive-hemagglutination technique of Gold and Fudenberg (10) for serological diagnosis of this condition. The test is reliable, specific, and sensitive and yields reproducible results.
MATERIALS AND METHODS Patients. Patients with severe, recurrent clinical vaginal candidiasis and with different degrees of infection were referred from the Obstetrics and Gynecology Department at the Medical University of South Carolina. Normal samples from adult Americans were obtained from the Family Practice Clinic at the Medical University of South Carolina. Normal samples from the Finiish population were obtained from blood donors of the Finnish Red Cross Blood Transfusion Service, Helsinki, Finland. Antigens. C. albicans and Cryptococcus neoformans were isolated from infected patients and maintained on synthetic agar medium at room temperature. For preparation of antigens, the fungi were transferred from the stock culture to liquid medium containing yeast nitrogen base and 1% dextrose and incubated for 48 h. The cells were harvested, inactivated, and washed three times with physiological saline. After washing, the cells were broken in a Braun homogenizer for 10 min. The clear supernatant was collected and dialyzed against saline for 24 h with three changes of physiological saline. The dialyzed solution was then filtered through Sephadex G-75 using tris(hydroxymethyl)aminothane(Tris)-hydrochloride buffer, pH 8.7. The fractions were pooled, concentrated, and analyzed by sufficiently systematized. sodium dodecyl sulfate-polyacrylamide gel electro' This is publication no. 68 from the Department of Basic phoresis. The first peak eluted was found to be rich in protein antigens and was used to coat the erythroand Clinical Immunology and Microbiology, Medical Unicytes. versity of South Carolina, Charleston, S.C. 287
288
Antisera. Rabbit sera hyperimmune to C. albiand C. neoformans were obtained from the Clinical Mycology Laboratory of the Medical University of South Carolina. Antisera for detection of various immunoglobulins were obtained from Burroughs Wellcome Co. Antiserum to secretary component was obtained from Behring Diagnostic, Somerville, N.J. The specificity of the antiserum was checked with proper controls. Fluorescein-conjugated sheep antisera to human immunoglobulin G (IgG), IgA, and IgM were prepared by Maria F. Lopez-Virella. The specificity of the antisera was thoroughly checked before using by double immunodiffusion against pure IgG, IgA, and IgM proteins. Techniques. (i) Passive hemagglutination. Candida and Cryptococcus antigens were coupled on 0 Rh-positive erythrocytes with chromic chloride (10). The best coating was obtained with 0.05% chromic chloride and a 1.5-mg/ml concentration of antigen. A 0.2 to 0.5% suspension of the coated erythrocytes was prepared in 0.15 M saline containing 0. 2% gelatin. The test was carried out in microtiter plates (Cooke Co.) with V-bottom wells in which serial dilutions of serum samples, starting at 1:2 dilution, were made. Pooled human serum absorbed with the antigen and an individual serum negative for antiCandida antibodies served as a negative control. (ii) Double diffusion. The double-diffusion technique (17) with 1% agar in 0.3 M KH2PO4 buffer at pH 8.0 was employed for the detection of precipitins to Candida in the sera of women with vaginal candidiasis, as well as in the determination of immunoglobulins in the fractions of these sera. Results were read after washing and staining of the plates with amido black (Bio-Rad). (iii) Indirect fluorescent-antibody technique. The indirect fluorescent-antibody technique, as described by Taschdjian et al. (21), was employed for determination of the nature of anti-Candida antibodies in the sera of systemic and vaginal candidiasis patients, as well as fractions of three sera of vaginal candidiasis patients. Clean glass slides were coated uniformly with whole cells of Candida in circular areas of 15-mm diameter. Serial dilutions of experimental sera were applied on the antigen smears, incubated for 30 min, and rinsed with phosphate-buffered saline (pH 7.3) and distilled water. The slides were air-dried, and drops of fluorescein isothiocyanate-conjugated sheep anti-human IgG, IgA, and IgM were applied on the circles and incubated for 30 min. The slides were then washed in phosphate-buffered saline and distilled water and air-dried. The smears were examined with a Leitz "Igniter" with DK condenser, filters KG-1 and BG38, and filter shield K-460. The light source was a Universal lamp 250A-BO-200. The last serum dilution to reveal Candida spores in at least partially fluorescent outline was considered the end point. (iv) Gel filtration. Sera of vaginal candidiasis patients were fractionated using Bio-Gel A 1.5m (Bio-Rad) and Ultragel ACA 34 (LKB). The buffer used for eluting the fractions was Tris-Tris-hydrochloride buffer, 0.2 M, pH 8.7. cans
INFECT. IMMUN.
MATHUR ET AL.
RESULTS
Serum antibodies to C. albicans. No antibodies to Candida were detected in the sera of vaginal candidiasis patients by double diffusion. By the passive-hemagglutination test, the antibody titers of vaginal candidiasis patients were significantly higher than those of the control population and lower than that of the systemic candidiasis patient (Table 1). The normal American population, consisting of 50 subjects, exhibited a range of 0 to 16 (arithmetic mean, 11) antibody titer to Candida. Ninety-eight normal Finnish subjects exhibited antibody titers ranging from 0 to 32 (mean, 10). Thirtyseven vaginal candidiasis patients had antibody titers ranging from 4 to 256 (mean, 55). The low titer values obtained in 3 of the 37 patients correlated with reduced infection after treatment or partial clinical remission, whereas in 2 patients, low titers could be observed even in the presence of clinically active infection. Cross-reactivity between C. albicans and C. neoformans. Erythrocytes coated with Candida and Cryptococcus were tested against serial dilutions of rabbit antisera and sera from 11 candidiasis and 9 cryptococcoid patients. Only slight cross-reactivity was found (Table 2).
Nature of antibodies to C. albicans in vaginal candidiasis. Sera of three vaginal candidiasis patients (B.L., J.M., and S.Mo.) were fractionated by gel filtration through Bio-Gel A 1.5m in the first case and Ultragel ACA 34 in the other two. A representative example of the elution profile (patient J.M.) is shown in Fig. 1. The immunoglobulins of different fractions were determined using the double-diffusion technique, and the fractions were pooled according to their contents. The antibody titers of TABLE 1. Antibody titers" (expressed as arithmetic mean) of vaginal candidiasis patients and controls, determined by passive hemagglutination and double diffusion Agglutinin tiSource of sample Normal American population Normal Finnish blood donors Vaginal candidiasis patients Systemic candidiasis patient
No. of subjects 50
ter (recipro- Precipical) tin present Mean Range 11 0-16 0
98
10
0-32
0
37
55
4-256
0
1
266
+
'Antibody titers were significantly higher for candidiasis patients than for controls (P < 0.01, Rank's test).
VOL. 15, 1977
TABLE 2. Cross-reactivity between Candida albicans and Cryptococcus neoformans Reciprocals of antibody titers against erythrocytes coated with antigen from:
Sample Rabbit antiserum to C. albicans Rabbit antiserum to C. neoformans Healthy control (male) Systemic candidiasis patients Vaginal candidiasis patients L.M. W.C. J.M. B.L. P.L. W.C. S.Ma. L.P. S.Mo. S.W. Cryptococcoid patients F.C. W.L. B.H. W.M. M.G. W.H. B.T. W.T. G.E.
Candida 554
Cryptococcus 11
120
526
5
9
266
1
9
5
5 154 21 256 149 3 75
1
3 4 3 1 1
11
6 6
75
0
1
0
390 80 49
146
101
0 0 0 0 0
45 5 2
47
fractions containing IgA and IgM to detect the presence of secretary component. All fractions that were rich in IgA and produced significant antibody titers to C. albicans were also found to contain secretary component (Fig. 2 and 3). Similar results were obtained with the indirect fluorescent-antibody technique. Whereas the systemic and mucocutaneous candidiasis patients M.W. and F.S. showed high fluorescence titers with antisera to the major immunoglobulins, the vaginal candidiasis patients showed only IgA anti-Candida antibodies (Table 4, Fig. 4). The IgA-rich fractions of the three vaginal candidiasis sera produced elevated titers of fluorescent anti-Candida antibody. Fractions containing IgG and IgM showed hardly any fluorescent antibody to Candida (Fig. 5). No fluorescence was observed when the sera from vaginal candidiasis patients were preabsorbed with C. albicans or with antiserum to IgA. The negative normal sera and human pool sera absorbed with C. albicans also showed negative fluorescence and were used as negative controls.
DISCUSSION The sensitivity of passive-hemagglutination methods for the detection of antibodies to C. albicans in cervicovaginal secretions of patients with vaginal candidiasis was first mentioned by Waldman et al. (24). The present studies support their findings. The successful use of the chromic chloride technique for coating erythrocytes is of practical importance, since it is easy, readily standardizable, reproducible, and specific in antibody determinations. Due to the ubiquitous nature of C. albicans, normal healthy subjects without clinical symp-
1 1
/00 80
W 60 I., 2 Oa 40, 0 "
289
HUMORAL IMMUNITY IN VAGINAL CANDIDIASIS
20
TABLE 3. Antibody titers in the fractions of sera from three patients with vaginal candidiasis 40
38
J36
34 32 30 28 26 FRACTI/ON NUM8ER
24
22
20
/5
FIG. 1. Elution profile of a serum sample from patient J.M., separated on an Ultragel ACA 34 column. Fractions 24 to 26 were pooled and found to contain IgA, secretary component, and anti-Candida antibody activity.
the pooled fractions of the three sera are shown in Table 3. Antibody activity was noted in fractions containing IgA with traces of other immunoglobulins. Maximum titers were obtained in fractions in which IgA was the only detectable immunoglobulin. Fractions containing IgG and IgM without IgA had no antibody activity. Double-diffusion tests were carried out on several
Pa-
Pea-t
Antibody titer of whole serum
Fraction
haracteriza-
(recip- No.
tion
rocal) B.L.
21
J.M.
154
58-62 63-98 21-23 24-26 27-29 30-31
S.Mo. a
11
32-37 20-22 23-27 28-33
IgM, IgA, SCa Other than IgA IgM and IgA IgA, SC IgA and traces of IgG IgA and traces of IgG IgG IgM IgM, IgA, SC IgG
SC, Secretory component.
Antibody titerocal) rcl 68 0 15 49 10 0.5 0 0 8 0
290
INFECT. IMMUN.
MATHUR ET AL.
MA
MAB
f'N
M
ME
AM D
MC
A
(Aib
FIG. 2. Characterization of pooled fractions of a serum sample from patient J.M. by immunodiffusion. Antisera specific for human IgG (G), IgA (A), IgM (M), and albumin (Alb) were used in the central wells. MA, Fractions 21-23; MB, 24-26; MC, 27-29; MD, 30-31; ME, 32-34; MF, 35-37.
toms carry circulating antibodies in serum with titers from 0 to 16 (Table 1). This is in accordance with the reports of Dolan and Stried (6) and Lehner (15, 16), who demonstrated antibody titers of 8 to 16 in normal subjects. In the current investigation, sera of 37 vaginal candidiasis patients showed antibody titers ranging from 3 to 256, with a mean of 55. Obviously, significantly elevated levels of serum antibodies could not be detected in all the patients with vaginal candidiasis. Thus, there were two groups of patients showing low, nondiagnostic titers - (i) three with regressing infection after adequate treatment and (ii) two with clinically active infection and paradoxically low antibody levels. Similar low antibody titers were also obtained by Waldman et al. (24). In the first group, there probably is a correlation between low disease activity and low antibody levels. The second group may be immune deficient, a possibility that deserves further investigation. In any case, confirmatory serological evidence for candidiasis was obtained in 35 out of 37
investigated patients, and this seems highly satisfactory. The antibodies detected in our studies are not totally specific for C. albicans, as proved in the studies of cross-reactivity with C. neoformans (Table 2). This may have been due to the inclusion of capsular polysaccharide antigens in the antigenic fraction used for coating the erythrocytes, since these are known to cross-react with Cryptococcus antigen (9; J. A. Faux, Ph.D. thesis, University of London, London, 1972). However, this is not a problem of clinical relevance, since the clinical pictures of candidiasis and cryptococcosis are not likely to be confused. In 1970, Lehner (16) attempted to determine the nature of antibodies to C. albicans in the serum of a systemic candidiasis patient. He concluded that the antibody population included IgG, IgA, and IgM molecules, with a predominance of IgG. In our studies with the fluorescent-antibody technique, we obtained similar results in the sera of systemic and mucocutaneous candidiasis patients. In the case of
VOL. 15, 1977
HUMORAL IMMUNITY IN VAGINAL CANDIDIASIS
.tjoooilowmv
291
N I
SA
8/6 2 55
ISc MC
SB
MB FIG. 3. Immunodiffusion test for the presence of secretary component in IgA fractions of three sera from patients with vaginal candidiasis. Lines of identity with the colostrum control (COL) against antiserum to secretary component (SC) reveal the presence of secretary component in fractions 58-62 ofpatient BL., 24-26 (MB) of patient J.M., and 23-27 (SA) of patient S.Mo. The contents of the other wells are as in Fig. 2.
vaginal candidiasis, however, anti-Candida antibodies seem restricted to the IgA class (Table 4, Fig. 4 and 5). Furthermore, the fractionation of sera from the three vaginal candidiasis patients (Table 3, Fig. 3) indicated that the circulating IgA antibody was of high molecular weight and contained secretary component (i.e., it could be defined as secretary IgA). Waldman et al. (24) reported the existence of secretary IgA in cervicovaginal secretions and the IgA nature of antibodies to C. albicans. Thus, the detection of secretary IgA antibodies to C. albicans in the sera of patients with vaginal candidiasis not only confirms the importance of local stimulation and regional response in mucocutaneous infections (11, 22, 23, 25), but also raises important considerations as to the possibility of a significant back-flow into systemic circulation of antibodies produced by perimucosal immunocytes. An alternative possibility would be the reabsorption of secreted antibody due to local inflammation and increased vascular permeability. This mechanism of
reabsorption was favored by Brandtzaeg (3) in his explanation of the increase ofpolymeric IgA with secretary component in the sera of patients with intestinal disorders. Our findings, although suggesting such a possibility, do not allow any definite conclusion about the mechanism responsible for the presence of secretary IgA antibodies to C. albicans in the sera of vaginal candidiasis patients. If reabsorption were the origin of this secretary antibody, a correlation between titer and intensity of inflammation could be expected; but other factors, such as the immune response capacity of the host, could complicate this pattern. The method of passive hemagglutination, using chromic chloride as coupling agent, has proven to be a sensitive tool for such studies. ACKNOWLEDGMENTS This research was supported by Public Health Service grant AI-13484 from the National Institute of Allergy and Infectious Diseases. We are grateful to R. Boackle for providing the antiserum for secretary component, to Maria F. Lopez-Virella for
A
B
C
D FIG. 4
w
A
B
E FIG. 5 292
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HUMORAL IMMUNITY IN VAGINAL CANDIDIASIS
TABLE 4. Titers offluorescent ayantibodies to C. albicans in sera from patients Lvith systemic, mucocutaneous, or vaginal candid asis and in serum fractions from three vaginal canid,lidiasis patients Fluorescent-anti-
body titer
Sample IgG
IgA
Normal
2 64
4 64
IgM 4 16
Mucocutaneous (F.S.)
32
64
32
32 16
0 0
A.W. E.Y. B.L. Whole serum Fractions 58-62
0 0 0 0 0 0
32
0 0
64 32
Fractions 63-98
0
0
0 0 0 0 0
64 16 32 8 0
subjects Systemic (M.W.)
Vaginal L.M. S.Ma. R.W.
E.C.
J.M. Whole serum Fractions 21-23 Fractions 24-26
Fractions 27-29 Fractions 30-31 Fractions 32-37
8 64
64
0
0 0 0
0 0 0 0 0 0
0
0
0 0 0
S.Mo. Fractions 20-22 Fractions 23-27 Fractions 28-33
0 00 0
0 16 0
06
0 00 0
L.M. Absorbed with C. albicans Absorbed with anti-IgA
0 0
0 0
0 0
the preparation of fluoresceip-conjugat;ed antisera to IgG, IgA, and IgM, and to Nancy Sack for aAssistance in obtaining sera from patients. We are thanksful for the editorial assistance by Charles Smith and typo;graphical help rendered by Heidi Gathright. LITERATURE CITE:D 1. Anderson, P. L., and A. Stenderup. 1974. Candida albicans antibodies in candidiasis. S5 cand. J. Infect. Dis. 6:69-73. 2. Axelson, N. H., C. H. Kirckpatrick, and R. H. Buckley. 1974. Precipitins to Candida albiocans in chronic mucocutaneous candidiasis studied by crossed immunoe-
lectrophoresis with intermediat;e gel. Correlation with clinical and immunological findings. Clin. Exp. Immunol. 17:385-394. 3. Brandtzaeg, P. 1971. Human secre-tory immunoglobulins. V. Occurrence of secretary piece in human serum. J. Immunol. 106:318-323. 4. Brody, J. I., and S. L. Finch. 196(D. Candida-reacting
293
antibody in the serum of patients with lymphomas and related disorders. Blood 15:830-834. 5. Buck, A. A., and H. F. Hasenclever. 1963. Epidemiological studies of skin reactions and serum agglutinins to Candida albicans in pregnant women. Am. J. Hyg. 78:232-240.
6. Dolan, C. T., and R. P. Stried. 1973. Serologic diagnosis of yeast infections. Am. J. Clin. Pathol. 59:49-55. 7. Esterly, N. B. 1968. Serum antibody titers to Candida albicans utilizing an immunofluorescent technique.
Am. J. Clin. Pathol. 50:291-296. 8. Evans, E. G. V., and K. T. Holland. 1975. Action of Candida precipitins. A comparison of double diffusion and counterimmunoelectrophoresis. J. Immunol. Methods 7:211-218. 9. Evans, E. E., L. J. Sorenson, of K. W. Walls. 1953. The antigenic composition andCryptococcus neoformans. V. A survey of cross-reactions among strains of Cryptococcus and other antigens. J. Bacteriol. 66:287-293. 10. Gold, E. R., and H. H. Fudenberg. 1967. Chromic chloride: a coupling reagent for passive hemagglutination reactions. J. Immunol. 99:859-866. J. F. 1975. The secretary immune system. A 11. Heremans, critical appraisal, p. 376-385. In The immune system and infectious diseases, Fourth International Convocation on Immunology, Buffalo, N.Y. Karger, Basel. 12. Holti, G. 1966. Candida allergy, p. 73-81. In Symposium on Candida infections. S. Livingstone, Edinburgh and London. H. I. Winner and R. E. Hurley (ed.). 13. Kaplan, W. 1966. Practical application of fluorescent antibody procedures in medical mycology. Pub. Pan Am. Health Org. 304:178-186. 14. Kozinn, P. J., J. Lynfield, and M. S. Seeling. 1974. Successful treatment of systemic candidiasis following cardiac surgery. Am. J. Dis. Child. 128:106-108. 15. Lehner, T. 1965. Immunofluorescent investigation of Candida albicans antibodies in human saliva. Arch. Oral Biol. 10:975-980. 16. Lehner, T. 1970. Serum fluorescent antibody and immunoglobulin estimations in candidosis. J. Med. Microbiol. 3:475-481. 17. Outcherlony, O., and L. A. Nilsson. 1973. Immunodiffusion and immunoelectrophoresis. In D. M. Weir (ed.), Handbook of experimental immunology, vol. 1. Immunochemistry. Blackwell Scientific Publications, Oxford. 18. Preisler, H. D., H. F. Hasenclever, and A. Levitan. 1969. Serologic diagnosis of disseminated candidiasis in patients with acute leukemia. Ann. Intern. Med. 70:19-30. 19. Rosner, F., G. Gabriel, C. L. Taschdjian, M. B. Cuesta, and P. J. Kozinn. 1971. Serologic diagnosis of systemic candidiasis in patients with acute leukemia. Am. J. Med. 51:54-62. 20. Stanley, V. C., and R. Hurley. 1974. Candida precipitins in pregnant women: validity of the test systems used. J. Clin. Pathol. 27:66-69. 21. Taschdjian, C. L., P. J. Kozinn, M. B. Cuesta, and E. F. Toni. 1972. Serodiagnostic findings in proven candidal infections. Am. J. Clin. Pathol. 57:195-205. 22. Tomasi, T. B., Jr. 1972. Secretory immunoglobulins. N. Engl. J. Med. 287:500-506. 23. Vaerman, J. P., and J. Ferrin. 1974. Local immunologi-
FIG. 4. Spores of C. albicans showing the presence of IgA anti-Candida antibodies after treatment with serum from vaginal candidiasis patient L.M. (A). Negative fluorescence was obtained when the serum was treated with fluorescein-conjugated anti-IgG (B) or IgM (C) or absorbed with C. albicans (D). FIG. 5. Spores of C. albicans showing the presence offluorescentIgA anti-Candida antibodies in the serum (W) of vaginal candidiasis patient J.M. and its fractions 21-23 (A) and 24-26 (B). Negative fluoresence was
obtained with other fractions (E).
294
MATHUR ET AL.
cal response in the vagina, cervix and endometrium, p. 281-285. In Karolinska Symposia on Research Methods in Reproductive Endocrinology, 7th Symposium: immunological approaches to fertility control. Bogtrykkeriet Forum, Copenhagen. 24. Waldman, R. H., J. M. Cruz, and D. S. Rowe. 1971. Immunoglobulin levels and antibody to Candida albicans in human cervicovaginal secretions. Clin. Exp. Immunol. 9:427-434.
INFECT. IMMUN. 25. Waldman, R. H., and R. Ganguly. 1974. Techniques for eliciting mucosal immune response, p. 262-280. In Karolinska Symposia on Research Methods in Reproductive Endocrinology, 7th Symposium: immunological approaches to fertility control. Bogtrykkeriet Forum, Copenhagen. 26. Winner, H. I. 1955. A study of Candida albicans agglutinins in human sera. J. Hyg. 53:509-512.
INFECTION AND IMMUNITY, Jan. 1977, p. 287-294 Copyright ©) 1977 American Society for Microbiology
Humoral Immunity in Vaginal Candidiasis1 SUBBI MATHUR,* G. VIRELLA, J. KOISTINEN, E. 0. HORGER III, T. A. MAHVI, AND H. HUGH FUDENBERG Department of Basic and Clinical Immunology and Microbiology,* and Department of Obstetrics and Gynecology, Medical University of South Carolina, Charleston, South Carolina 29401 Received for publication 9 September 1976
Serum antibody titers to Candida albicans were estimated in 37 women with recurrent vaginal candidiasis and in 148 normal American and Finnish subjects, using the passive-hemagglutination technique. The antibody titers ranged from 0 to 16 in normal individuals and 4 to 256 in vaginal candidiasis patients. Antibodies to C. albicans in the sera of vaginal candidiasis patients were found to be the secretary immunoglobulin A type, as determined by gel filtration and double-diffusion tests. The results were confirmed by the indirect fluorescentantibody technique. Our findings suggest that, in vaginal candidiasis, the antibody response is mainly local, consisting of secretary immunoglobulin A, some of which finds its way into systemic circulation.
Candida albicans, a saprophytic fungus belonging to the Cryptococcaceae, is a common constituent of the microflora in body cavities. However, it assumes the role of a severe pathogen under certain circumstances, such as malignancies (4, 16, 18, 19, 20), major surgery involving the use of immunosuppressants and broad-spectrum antibiotics (14), or pregnancy (5). In women, vaginal candidiasis is a common and chronic ailment. Since this fungus is a normal inhabitant of the vaginal area, microscope evidence alone may not suffice for detection of active infection. An effective serodiagnostic tool would therefore be useful and would allow some insight into the patient's humoral immune response to the fungus. Tube agglutination (1), immunodiffusion (6, 8, 18, 19, 21), counterimmunoelectrophoresis (2, 8), and indirect fluorescent-antibody techniques (6, 7, 13, 15, 21) have been used, largely in studies on systemic candidiasis. Precipitin techniques yield positive results in 89 to 95% of cases (8, 14, 21). Agglutination techniques have been demonstrated to be more sensitive, and positive precipitins by double diffusion usually correspond to agglutination titers of 1:60 to 1:256 (1, 18, 19). There is little information available, however, in regard to the humoral immunity of vaginal candidiasis patients. Double diffusion has been shown to be insufficiently sensitive (20), and previous studies with agglutination methods (5, 12, 24, 26) have not been
The present paper describes the characterization of circulating antibodies in patients with vaginal candidiasis and the application of the passive-hemagglutination technique of Gold and Fudenberg (10) for serological diagnosis of this condition. The test is reliable, specific, and sensitive and yields reproducible results.
MATERIALS AND METHODS Patients. Patients with severe, recurrent clinical vaginal candidiasis and with different degrees of infection were referred from the Obstetrics and Gynecology Department at the Medical University of South Carolina. Normal samples from adult Americans were obtained from the Family Practice Clinic at the Medical University of South Carolina. Normal samples from the Finiish population were obtained from blood donors of the Finnish Red Cross Blood Transfusion Service, Helsinki, Finland. Antigens. C. albicans and Cryptococcus neoformans were isolated from infected patients and maintained on synthetic agar medium at room temperature. For preparation of antigens, the fungi were transferred from the stock culture to liquid medium containing yeast nitrogen base and 1% dextrose and incubated for 48 h. The cells were harvested, inactivated, and washed three times with physiological saline. After washing, the cells were broken in a Braun homogenizer for 10 min. The clear supernatant was collected and dialyzed against saline for 24 h with three changes of physiological saline. The dialyzed solution was then filtered through Sephadex G-75 using tris(hydroxymethyl)aminothane(Tris)-hydrochloride buffer, pH 8.7. The fractions were pooled, concentrated, and analyzed by sufficiently systematized. sodium dodecyl sulfate-polyacrylamide gel electro' This is publication no. 68 from the Department of Basic phoresis. The first peak eluted was found to be rich in protein antigens and was used to coat the erythroand Clinical Immunology and Microbiology, Medical Unicytes. versity of South Carolina, Charleston, S.C. 287
288
Antisera. Rabbit sera hyperimmune to C. albiand C. neoformans were obtained from the Clinical Mycology Laboratory of the Medical University of South Carolina. Antisera for detection of various immunoglobulins were obtained from Burroughs Wellcome Co. Antiserum to secretary component was obtained from Behring Diagnostic, Somerville, N.J. The specificity of the antiserum was checked with proper controls. Fluorescein-conjugated sheep antisera to human immunoglobulin G (IgG), IgA, and IgM were prepared by Maria F. Lopez-Virella. The specificity of the antisera was thoroughly checked before using by double immunodiffusion against pure IgG, IgA, and IgM proteins. Techniques. (i) Passive hemagglutination. Candida and Cryptococcus antigens were coupled on 0 Rh-positive erythrocytes with chromic chloride (10). The best coating was obtained with 0.05% chromic chloride and a 1.5-mg/ml concentration of antigen. A 0.2 to 0.5% suspension of the coated erythrocytes was prepared in 0.15 M saline containing 0. 2% gelatin. The test was carried out in microtiter plates (Cooke Co.) with V-bottom wells in which serial dilutions of serum samples, starting at 1:2 dilution, were made. Pooled human serum absorbed with the antigen and an individual serum negative for antiCandida antibodies served as a negative control. (ii) Double diffusion. The double-diffusion technique (17) with 1% agar in 0.3 M KH2PO4 buffer at pH 8.0 was employed for the detection of precipitins to Candida in the sera of women with vaginal candidiasis, as well as in the determination of immunoglobulins in the fractions of these sera. Results were read after washing and staining of the plates with amido black (Bio-Rad). (iii) Indirect fluorescent-antibody technique. The indirect fluorescent-antibody technique, as described by Taschdjian et al. (21), was employed for determination of the nature of anti-Candida antibodies in the sera of systemic and vaginal candidiasis patients, as well as fractions of three sera of vaginal candidiasis patients. Clean glass slides were coated uniformly with whole cells of Candida in circular areas of 15-mm diameter. Serial dilutions of experimental sera were applied on the antigen smears, incubated for 30 min, and rinsed with phosphate-buffered saline (pH 7.3) and distilled water. The slides were air-dried, and drops of fluorescein isothiocyanate-conjugated sheep anti-human IgG, IgA, and IgM were applied on the circles and incubated for 30 min. The slides were then washed in phosphate-buffered saline and distilled water and air-dried. The smears were examined with a Leitz "Igniter" with DK condenser, filters KG-1 and BG38, and filter shield K-460. The light source was a Universal lamp 250A-BO-200. The last serum dilution to reveal Candida spores in at least partially fluorescent outline was considered the end point. (iv) Gel filtration. Sera of vaginal candidiasis patients were fractionated using Bio-Gel A 1.5m (Bio-Rad) and Ultragel ACA 34 (LKB). The buffer used for eluting the fractions was Tris-Tris-hydrochloride buffer, 0.2 M, pH 8.7. cans
INFECT. IMMUN.
MATHUR ET AL.
RESULTS
Serum antibodies to C. albicans. No antibodies to Candida were detected in the sera of vaginal candidiasis patients by double diffusion. By the passive-hemagglutination test, the antibody titers of vaginal candidiasis patients were significantly higher than those of the control population and lower than that of the systemic candidiasis patient (Table 1). The normal American population, consisting of 50 subjects, exhibited a range of 0 to 16 (arithmetic mean, 11) antibody titer to Candida. Ninety-eight normal Finnish subjects exhibited antibody titers ranging from 0 to 32 (mean, 10). Thirtyseven vaginal candidiasis patients had antibody titers ranging from 4 to 256 (mean, 55). The low titer values obtained in 3 of the 37 patients correlated with reduced infection after treatment or partial clinical remission, whereas in 2 patients, low titers could be observed even in the presence of clinically active infection. Cross-reactivity between C. albicans and C. neoformans. Erythrocytes coated with Candida and Cryptococcus were tested against serial dilutions of rabbit antisera and sera from 11 candidiasis and 9 cryptococcoid patients. Only slight cross-reactivity was found (Table 2).
Nature of antibodies to C. albicans in vaginal candidiasis. Sera of three vaginal candidiasis patients (B.L., J.M., and S.Mo.) were fractionated by gel filtration through Bio-Gel A 1.5m in the first case and Ultragel ACA 34 in the other two. A representative example of the elution profile (patient J.M.) is shown in Fig. 1. The immunoglobulins of different fractions were determined using the double-diffusion technique, and the fractions were pooled according to their contents. The antibody titers of TABLE 1. Antibody titers" (expressed as arithmetic mean) of vaginal candidiasis patients and controls, determined by passive hemagglutination and double diffusion Agglutinin tiSource of sample Normal American population Normal Finnish blood donors Vaginal candidiasis patients Systemic candidiasis patient
No. of subjects 50
ter (recipro- Precipical) tin present Mean Range 11 0-16 0
98
10
0-32
0
37
55
4-256
0
1
266
+
'Antibody titers were significantly higher for candidiasis patients than for controls (P < 0.01, Rank's test).
VOL. 15, 1977
TABLE 2. Cross-reactivity between Candida albicans and Cryptococcus neoformans Reciprocals of antibody titers against erythrocytes coated with antigen from:
Sample Rabbit antiserum to C. albicans Rabbit antiserum to C. neoformans Healthy control (male) Systemic candidiasis patients Vaginal candidiasis patients L.M. W.C. J.M. B.L. P.L. W.C. S.Ma. L.P. S.Mo. S.W. Cryptococcoid patients F.C. W.L. B.H. W.M. M.G. W.H. B.T. W.T. G.E.
Candida 554
Cryptococcus 11
120
526
5
9
266
1
9
5
5 154 21 256 149 3 75
1
3 4 3 1 1
11
6 6
75
0
1
0
390 80 49
146
101
0 0 0 0 0
45 5 2
47
fractions containing IgA and IgM to detect the presence of secretary component. All fractions that were rich in IgA and produced significant antibody titers to C. albicans were also found to contain secretary component (Fig. 2 and 3). Similar results were obtained with the indirect fluorescent-antibody technique. Whereas the systemic and mucocutaneous candidiasis patients M.W. and F.S. showed high fluorescence titers with antisera to the major immunoglobulins, the vaginal candidiasis patients showed only IgA anti-Candida antibodies (Table 4, Fig. 4). The IgA-rich fractions of the three vaginal candidiasis sera produced elevated titers of fluorescent anti-Candida antibody. Fractions containing IgG and IgM showed hardly any fluorescent antibody to Candida (Fig. 5). No fluorescence was observed when the sera from vaginal candidiasis patients were preabsorbed with C. albicans or with antiserum to IgA. The negative normal sera and human pool sera absorbed with C. albicans also showed negative fluorescence and were used as negative controls.
DISCUSSION The sensitivity of passive-hemagglutination methods for the detection of antibodies to C. albicans in cervicovaginal secretions of patients with vaginal candidiasis was first mentioned by Waldman et al. (24). The present studies support their findings. The successful use of the chromic chloride technique for coating erythrocytes is of practical importance, since it is easy, readily standardizable, reproducible, and specific in antibody determinations. Due to the ubiquitous nature of C. albicans, normal healthy subjects without clinical symp-
1 1
/00 80
W 60 I., 2 Oa 40, 0 "
289
HUMORAL IMMUNITY IN VAGINAL CANDIDIASIS
20
TABLE 3. Antibody titers in the fractions of sera from three patients with vaginal candidiasis 40
38
J36
34 32 30 28 26 FRACTI/ON NUM8ER
24
22
20
/5
FIG. 1. Elution profile of a serum sample from patient J.M., separated on an Ultragel ACA 34 column. Fractions 24 to 26 were pooled and found to contain IgA, secretary component, and anti-Candida antibody activity.
the pooled fractions of the three sera are shown in Table 3. Antibody activity was noted in fractions containing IgA with traces of other immunoglobulins. Maximum titers were obtained in fractions in which IgA was the only detectable immunoglobulin. Fractions containing IgG and IgM without IgA had no antibody activity. Double-diffusion tests were carried out on several
Pa-
Pea-t
Antibody titer of whole serum
Fraction
haracteriza-
(recip- No.
tion
rocal) B.L.
21
J.M.
154
58-62 63-98 21-23 24-26 27-29 30-31
S.Mo. a
11
32-37 20-22 23-27 28-33
IgM, IgA, SCa Other than IgA IgM and IgA IgA, SC IgA and traces of IgG IgA and traces of IgG IgG IgM IgM, IgA, SC IgG
SC, Secretory component.
Antibody titerocal) rcl 68 0 15 49 10 0.5 0 0 8 0
290
INFECT. IMMUN.
MATHUR ET AL.
MA
MAB
f'N
M
ME
AM D
MC
A
(Aib
FIG. 2. Characterization of pooled fractions of a serum sample from patient J.M. by immunodiffusion. Antisera specific for human IgG (G), IgA (A), IgM (M), and albumin (Alb) were used in the central wells. MA, Fractions 21-23; MB, 24-26; MC, 27-29; MD, 30-31; ME, 32-34; MF, 35-37.
toms carry circulating antibodies in serum with titers from 0 to 16 (Table 1). This is in accordance with the reports of Dolan and Stried (6) and Lehner (15, 16), who demonstrated antibody titers of 8 to 16 in normal subjects. In the current investigation, sera of 37 vaginal candidiasis patients showed antibody titers ranging from 3 to 256, with a mean of 55. Obviously, significantly elevated levels of serum antibodies could not be detected in all the patients with vaginal candidiasis. Thus, there were two groups of patients showing low, nondiagnostic titers - (i) three with regressing infection after adequate treatment and (ii) two with clinically active infection and paradoxically low antibody levels. Similar low antibody titers were also obtained by Waldman et al. (24). In the first group, there probably is a correlation between low disease activity and low antibody levels. The second group may be immune deficient, a possibility that deserves further investigation. In any case, confirmatory serological evidence for candidiasis was obtained in 35 out of 37
investigated patients, and this seems highly satisfactory. The antibodies detected in our studies are not totally specific for C. albicans, as proved in the studies of cross-reactivity with C. neoformans (Table 2). This may have been due to the inclusion of capsular polysaccharide antigens in the antigenic fraction used for coating the erythrocytes, since these are known to cross-react with Cryptococcus antigen (9; J. A. Faux, Ph.D. thesis, University of London, London, 1972). However, this is not a problem of clinical relevance, since the clinical pictures of candidiasis and cryptococcosis are not likely to be confused. In 1970, Lehner (16) attempted to determine the nature of antibodies to C. albicans in the serum of a systemic candidiasis patient. He concluded that the antibody population included IgG, IgA, and IgM molecules, with a predominance of IgG. In our studies with the fluorescent-antibody technique, we obtained similar results in the sera of systemic and mucocutaneous candidiasis patients. In the case of
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HUMORAL IMMUNITY IN VAGINAL CANDIDIASIS
.tjoooilowmv
291
N I
SA
8/6 2 55
ISc MC
SB
MB FIG. 3. Immunodiffusion test for the presence of secretary component in IgA fractions of three sera from patients with vaginal candidiasis. Lines of identity with the colostrum control (COL) against antiserum to secretary component (SC) reveal the presence of secretary component in fractions 58-62 ofpatient BL., 24-26 (MB) of patient J.M., and 23-27 (SA) of patient S.Mo. The contents of the other wells are as in Fig. 2.
vaginal candidiasis, however, anti-Candida antibodies seem restricted to the IgA class (Table 4, Fig. 4 and 5). Furthermore, the fractionation of sera from the three vaginal candidiasis patients (Table 3, Fig. 3) indicated that the circulating IgA antibody was of high molecular weight and contained secretary component (i.e., it could be defined as secretary IgA). Waldman et al. (24) reported the existence of secretary IgA in cervicovaginal secretions and the IgA nature of antibodies to C. albicans. Thus, the detection of secretary IgA antibodies to C. albicans in the sera of patients with vaginal candidiasis not only confirms the importance of local stimulation and regional response in mucocutaneous infections (11, 22, 23, 25), but also raises important considerations as to the possibility of a significant back-flow into systemic circulation of antibodies produced by perimucosal immunocytes. An alternative possibility would be the reabsorption of secreted antibody due to local inflammation and increased vascular permeability. This mechanism of
reabsorption was favored by Brandtzaeg (3) in his explanation of the increase ofpolymeric IgA with secretary component in the sera of patients with intestinal disorders. Our findings, although suggesting such a possibility, do not allow any definite conclusion about the mechanism responsible for the presence of secretary IgA antibodies to C. albicans in the sera of vaginal candidiasis patients. If reabsorption were the origin of this secretary antibody, a correlation between titer and intensity of inflammation could be expected; but other factors, such as the immune response capacity of the host, could complicate this pattern. The method of passive hemagglutination, using chromic chloride as coupling agent, has proven to be a sensitive tool for such studies. ACKNOWLEDGMENTS This research was supported by Public Health Service grant AI-13484 from the National Institute of Allergy and Infectious Diseases. We are grateful to R. Boackle for providing the antiserum for secretary component, to Maria F. Lopez-Virella for
A
B
C
D FIG. 4
w
A
B
E FIG. 5 292
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HUMORAL IMMUNITY IN VAGINAL CANDIDIASIS
TABLE 4. Titers offluorescent ayantibodies to C. albicans in sera from patients Lvith systemic, mucocutaneous, or vaginal candid asis and in serum fractions from three vaginal canid,lidiasis patients Fluorescent-anti-
body titer
Sample IgG
IgA
Normal
2 64
4 64
IgM 4 16
Mucocutaneous (F.S.)
32
64
32
32 16
0 0
A.W. E.Y. B.L. Whole serum Fractions 58-62
0 0 0 0 0 0
32
0 0
64 32
Fractions 63-98
0
0
0 0 0 0 0
64 16 32 8 0
subjects Systemic (M.W.)
Vaginal L.M. S.Ma. R.W.
E.C.
J.M. Whole serum Fractions 21-23 Fractions 24-26
Fractions 27-29 Fractions 30-31 Fractions 32-37
8 64
64
0
0 0 0
0 0 0 0 0 0
0
0
0 0 0
S.Mo. Fractions 20-22 Fractions 23-27 Fractions 28-33
0 00 0
0 16 0
06
0 00 0
L.M. Absorbed with C. albicans Absorbed with anti-IgA
0 0
0 0
0 0
the preparation of fluoresceip-conjugat;ed antisera to IgG, IgA, and IgM, and to Nancy Sack for aAssistance in obtaining sera from patients. We are thanksful for the editorial assistance by Charles Smith and typo;graphical help rendered by Heidi Gathright. LITERATURE CITE:D 1. Anderson, P. L., and A. Stenderup. 1974. Candida albicans antibodies in candidiasis. S5 cand. J. Infect. Dis. 6:69-73. 2. Axelson, N. H., C. H. Kirckpatrick, and R. H. Buckley. 1974. Precipitins to Candida albiocans in chronic mucocutaneous candidiasis studied by crossed immunoe-
lectrophoresis with intermediat;e gel. Correlation with clinical and immunological findings. Clin. Exp. Immunol. 17:385-394. 3. Brandtzaeg, P. 1971. Human secre-tory immunoglobulins. V. Occurrence of secretary piece in human serum. J. Immunol. 106:318-323. 4. Brody, J. I., and S. L. Finch. 196(D. Candida-reacting
293
antibody in the serum of patients with lymphomas and related disorders. Blood 15:830-834. 5. Buck, A. A., and H. F. Hasenclever. 1963. Epidemiological studies of skin reactions and serum agglutinins to Candida albicans in pregnant women. Am. J. Hyg. 78:232-240.
6. Dolan, C. T., and R. P. Stried. 1973. Serologic diagnosis of yeast infections. Am. J. Clin. Pathol. 59:49-55. 7. Esterly, N. B. 1968. Serum antibody titers to Candida albicans utilizing an immunofluorescent technique.
Am. J. Clin. Pathol. 50:291-296. 8. Evans, E. G. V., and K. T. Holland. 1975. Action of Candida precipitins. A comparison of double diffusion and counterimmunoelectrophoresis. J. Immunol. Methods 7:211-218. 9. Evans, E. E., L. J. Sorenson, of K. W. Walls. 1953. The antigenic composition andCryptococcus neoformans. V. A survey of cross-reactions among strains of Cryptococcus and other antigens. J. Bacteriol. 66:287-293. 10. Gold, E. R., and H. H. Fudenberg. 1967. Chromic chloride: a coupling reagent for passive hemagglutination reactions. J. Immunol. 99:859-866. J. F. 1975. The secretary immune system. A 11. Heremans, critical appraisal, p. 376-385. In The immune system and infectious diseases, Fourth International Convocation on Immunology, Buffalo, N.Y. Karger, Basel. 12. Holti, G. 1966. Candida allergy, p. 73-81. In Symposium on Candida infections. S. Livingstone, Edinburgh and London. H. I. Winner and R. E. Hurley (ed.). 13. Kaplan, W. 1966. Practical application of fluorescent antibody procedures in medical mycology. Pub. Pan Am. Health Org. 304:178-186. 14. Kozinn, P. J., J. Lynfield, and M. S. Seeling. 1974. Successful treatment of systemic candidiasis following cardiac surgery. Am. J. Dis. Child. 128:106-108. 15. Lehner, T. 1965. Immunofluorescent investigation of Candida albicans antibodies in human saliva. Arch. Oral Biol. 10:975-980. 16. Lehner, T. 1970. Serum fluorescent antibody and immunoglobulin estimations in candidosis. J. Med. Microbiol. 3:475-481. 17. Outcherlony, O., and L. A. Nilsson. 1973. Immunodiffusion and immunoelectrophoresis. In D. M. Weir (ed.), Handbook of experimental immunology, vol. 1. Immunochemistry. Blackwell Scientific Publications, Oxford. 18. Preisler, H. D., H. F. Hasenclever, and A. Levitan. 1969. Serologic diagnosis of disseminated candidiasis in patients with acute leukemia. Ann. Intern. Med. 70:19-30. 19. Rosner, F., G. Gabriel, C. L. Taschdjian, M. B. Cuesta, and P. J. Kozinn. 1971. Serologic diagnosis of systemic candidiasis in patients with acute leukemia. Am. J. Med. 51:54-62. 20. Stanley, V. C., and R. Hurley. 1974. Candida precipitins in pregnant women: validity of the test systems used. J. Clin. Pathol. 27:66-69. 21. Taschdjian, C. L., P. J. Kozinn, M. B. Cuesta, and E. F. Toni. 1972. Serodiagnostic findings in proven candidal infections. Am. J. Clin. Pathol. 57:195-205. 22. Tomasi, T. B., Jr. 1972. Secretory immunoglobulins. N. Engl. J. Med. 287:500-506. 23. Vaerman, J. P., and J. Ferrin. 1974. Local immunologi-
FIG. 4. Spores of C. albicans showing the presence of IgA anti-Candida antibodies after treatment with serum from vaginal candidiasis patient L.M. (A). Negative fluorescence was obtained when the serum was treated with fluorescein-conjugated anti-IgG (B) or IgM (C) or absorbed with C. albicans (D). FIG. 5. Spores of C. albicans showing the presence offluorescentIgA anti-Candida antibodies in the serum (W) of vaginal candidiasis patient J.M. and its fractions 21-23 (A) and 24-26 (B). Negative fluoresence was
obtained with other fractions (E).
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cal response in the vagina, cervix and endometrium, p. 281-285. In Karolinska Symposia on Research Methods in Reproductive Endocrinology, 7th Symposium: immunological approaches to fertility control. Bogtrykkeriet Forum, Copenhagen. 24. Waldman, R. H., J. M. Cruz, and D. S. Rowe. 1971. Immunoglobulin levels and antibody to Candida albicans in human cervicovaginal secretions. Clin. Exp. Immunol. 9:427-434.
INFECT. IMMUN. 25. Waldman, R. H., and R. Ganguly. 1974. Techniques for eliciting mucosal immune response, p. 262-280. In Karolinska Symposia on Research Methods in Reproductive Endocrinology, 7th Symposium: immunological approaches to fertility control. Bogtrykkeriet Forum, Copenhagen. 26. Winner, H. I. 1955. A study of Candida albicans agglutinins in human sera. J. Hyg. 53:509-512.
