AKIBA, KAZUO IWATA AND SUTEMI INOUYE. Department of Bacteriology, Faculty of Medicine, University of Tokyo, Tokyo

Transcription

1 Japan. J. Microb., Vol. 1, No. 1, 1957 STUDIES ON THE SEROLOGIC DIAGNOSIS OF THE DEEP-SEATED CANDIDIASIS TOMOICHIRO AKIBA, KAZUO IWATA AND SUTEMI INOUYE Department of Bacteriology, Faculty of Medicine, University of Tokyo, Tokyo (Received October 22, 1956) The diagnosis of candidiasis is difficult, especially in such cases as deepseated infections without obvious clinical findings. Therefore, candidiasis must be diagnosed as such after performing clinical, mycological, serological and other related tests in order to ascertain it as Candida infection. In order to obtain an early diagnosis of candidiasis, the skin test is considered to be one of the most convenient methods. However, many investigators"' have reported that the skin test with vaccines or culture filtrates of Candida albicans (for example, monilien, oidiomycin, oidiomycetin, etc.) has little diagnostic value since a large percentage of the healthy individuals give positive reactions. Such shortcomings of the skin test may be attributed to the nature of the antigen used. Thus, in order to obtain a highly specific antigen for the skin test, precipitin reaction or other serologic reactions for candidiasis, an improved method was applied to the extraction of the antigenic substance from Candida albicans, and the value of its result was reexamined. At first, a polysaccharide fraction was extracted from the Candida cells. As a preliminary test, rabbits immunized or infected with Candida cells were tested for skin and precipitin reactions using the polysaccharide antigen. Since the result obtained was satisfactory, patients with candidiasis and various other diseases, along with healthy persons, were tested in a similar manner. EXPERIMENTAL METHODS AND RESULTS 1. Extraction and purification of polysaccharide fraction The procedures used were similar, in general, to Palmer and Gerlough's method.(2) C. albicans #2 isolated from the sputum of a patient with pulmonary candidiasis was cultivated on Sabouraud's glucose agar plate at 37 Ž for 3 days. The Candida cells were harvested and washed thoroughly with physiological saline solution, followed by 3 or 4 washings with acetone before drying in a dessicator. A 90-95% phenol solution was added to the dried cells and the suspension was held at 37 Ž for 2 to 3 days with occasional shaking. The suspension was filtered and the insoluble part was washed several times with ethanol to remove the r,esidual phenol. The sediment thus obtained was resuspended in distilled water and heated at 100 Ž for one hour. This suspen- sion was centrifuged and the supernatant dialyzed in running water. The dialyzed fraction was concentrated under reduced pressure and filtered

2 12 AKIBA, IWATA AND INOUYE Vol. 1, No. 1 through a I, Chamberland filter. Three volumes of absolute ethanol and sodium acetate (to make a final concentration of 1.0%) were added to the filtrate and held until precipitation occurred. The precipitate was dissolved once more in distilled water and dialyzed. After concentrating under reduced pressure, three volumes of absolute ethanol were added to precipitate the antigen. This procedure was repeated three times. The final precipitate was dried in a dessicator following washing with acetone. The average yield was about 50 mg per 10 g of dried cells. II. Chemical nature of the polysaccharide fraction The white powder obtained is easily soluble in water and thermostable withstanding 100 Ž heating for at least one hour without destruction. The chemical reactions are as follows : sugar reactions, Molisch reaction and carbazol reaction positive ; orcinol-hc1 reaction, phloroglucin reaction, ninhydrin reaction, Millon reaction and Hopkins-Cole reaction negative ; Sakaguchi reaction positive. These results show clearly that the material obtained is of polysaccharide nature. The results of quantitative chemical analysis were as follows : nitrogen, 1.42% ; phosphorus, not detected ; reducing sugar, 40.08%; acetyl, 5.91% ; and specific rotation, kƒ l= The polysaccharide fraction was hydrolyzed with 1N-H2SO for 3 hours. This solution was then chromatogrammed on filter paper with a butanolpyrimidine solution, and developed with ammonium-silver nitrate solution. The hydrolyzed fraction showed two spots ; the upper with a Rf value of 0.22 (similar to glucose control) and the lower with 0.04 (some hydrolysis-resistant substance). The Rf value of the latter was not identical with those of galactose, mannose and other reducing sugars which served as controls. Therefore, the main component of the reducing sugar which amounted to 40.08%, as described above, was considered to be glucose. For the ultraviolet absorption test, the antigen was dissolved in distilled Water and measured with a " Unicum " spectrophotometer. Maximum absorption was observed at about 255nip, and slightly at 270 mp and 330 mp. However, it is not clear whether these absorption maxima are specific or not. III. Experiments on rabbits immunized with dead cells of Candida The polysaccharide fraction was initially tested for its value as an antigen in skin test and precipitin reaction on rabbits immunized with dead cells of various Candida species. The strains used were C. albicans #2, C. albicans its, C. albicans,* C. tropicalis,* C. pseudotropicalis,* C. krusei,* C. parakrusei,* C. stellatoidea,* and C. guilliermondi.* The latter seven strains (*-marked) were sent to Japan by Dr. Conant of Duke University, U. S. A. The culture of Candida incubated at 37 Ž for 38 hours in Sabouraud's glucose broth was heat-killed (60 Ž for 30 minutes), and 0.5 to 2.0 cc of the suspension was inoculated intravenously into rabbits at definite intervals. During the course of immunization, the skin test and precipitin reaction were performed at various intervals using the polysaccharide solution. For the skin test, 0.05 cc of the antigen was injected intracutaneously (serially diluted in saline solution) into the shaved flank of rabbits at proper distance. The result of the reaction depended upon the appearance of reddening at sites of inocula-

3 _January 1957 SEROLOGIC DIAGNOSIS OF CANDIDIASIS 13 tion within 24 hours, with or without induration. The result was recorded as positive ( +), 10 mm or more ; doubtful positive ( }: ), 9-5 mm ; and negative ( - ), 4-0 mm in average diameter of reddness. The precipitin reaction was performed according to the usual ring test method, and the result was observed after holding the test tubes at room temperature for 2 hours. Agglutination reaction was performed by inhibiting the spontaneous agglutination. The culture of Candida incubated at 37 Ž for 24 hours on Sabouraud's glucose agar was harvested and washed twice with saline solution. The cells were suspended in saline solution and heated in a 60 Ž water bath for 30 minutes to kill the cells. Formalin was then added to bring the final concentration to 0.5%. A % saline solution was used for the suspension of cells and dilution of rabbit serum. A series of the mixtures of cell suspension and serum, after shaking, were held in a 50 Ž water bath for one hour. The results were observed and recorded after standing at room temperature for 24 hours. At first, in order to determine the relationship between the skin reaction and antibody titer, the above three reactions were tested on No. 2, No. 3 and " Duke " strains of C. albicans and their respective immunized rabbits, A to G. The skin reaction, in general, changed to positive and increased its intensity together with the rise in serum antibody titer. Similar results were also ob- Table 1. Relationship Between the Skin Sensitivity and the Titer of Serum Antibody Before and After Immunization of Rabbits with Killed Cells of C. albicans and Other Species

4 AKIBA, IWATA AND INOUYE Vol. 1, No. 1 taincd in all of the cases. Table 1 summarises such relationships, showing the titers of the reaction just before the first inoculation and those on the fifth day after the last, that is, 30 days after the first inoculation when the titers attained their maxima. Second, specificity of the antigen extracted from C. albicans, strain 2, was studied on rabbits immunized with killed cells of six different Candida species. The results are shown in the lower part of table 1. The polysaccharide fraction derived from C. albicans #2 showed considerable activity on rabbits immunized with C. tropicalis, while rabbits immunized with C. pseudotropicalis, C. krusei, C. parakrusei, C. steilatoidea and C. guilliermondi showed a slight cross reaction in the precipitin test, but without any recognizable increase in skin sensitivity. IV. Experiments on rabbits infected with living cells of Candida The rabbits infected with living Candida cells were tested for the same reactions. The test organisms, cultivated in Sabouraud's glucose broth at 37 Ž for 48 hours, were inoculated intraperitoneally or intravenously into a rabbit at a interval of 14 days. The challenge doses were 0.5 cc of homogenous cell suspension at the initial injection and 1 cc on reinjection. The resuls are shown in table 2. Likewise, as in the case of rabbits immunized with dead cells of Candida, the experiments on the animals undergoing systemic infection also showed a good parallel relationship between the degree of skin sensitivity and the titers of the precipitin reaction and, to some extent, of agglutination reaction. Table 2. Relationship Between the Skin Sensitivity and the Titer of Serum Antibody in Rabbits Infected with Candida sp. V. Preliminary investigations on patients with candidiasis, with related or unrelated diseases, and on healthy individuals Since the results of the animal experiments gave good evidence in estimating the antibody titer and the skin sensitivity of Candida infected persons, similar tests were performed on patients with candidiasis and, as their control, with several other diseases, and on healthy individuals. Male and female adults were selected at random. In this investigation, individuals tested were divided into two groups.

5 January 1957 SEROLOGIC DIAGNOSIS OF CANDIDIASIS 15 Group A included healthy individuals and patients who seemed to be apparently without candidiasis, for instance, acute appendicitis, etc. (111 cases). Group B included patients with candidiasis and related diseases ; 4 cases of deep-seated candidiasis, 70 cases of tuberculosis and 28 cases of various kinds of chronic diseases, for instance, lung carcinoma, diabetes, heart diseases, etc. (102 cases). The procedures for the foregoing tests were conducted in a manner similar to the experiments performed on the rabbits. Skin test. The inoculation site was the skin of the forearm. Table 3a Table 3a. Skin Test on Man Using Candida Polysaccharide Fraction (Preliminary Investigation) Table 3b. Precipitin Reaction in Man Using Candia Polysaccharide Fraction (Preliminary Investigation) Table 3c. Agglutination Reaction in Man (Preliminary Investigation)

6 16 AKIBA, IWATA AND INOUYE Vol. 1, No. 1 shows the result of this test. In group A, no positive reactions were observed when 0.1 fig or less of the antigen was injected, whereas in group B, 6 positive cases were observed with the same amount. Four of these cases were candidiasis and two tuberculosis. Precipitin reaction. As shown in table 3b, only one case in group A gave a positive reaction to a concentration of 250 Đg/cc of the antigen and none to 100 Đg/cc or less. In group B, positive reactions were observed in 7 cases with 250 Đg/cc, 4 cases with 100 Đg/cc, 2 cases with 50 Đg/cc or less. Four cases which give positive reactions to 100 Đg/cc or less were all candidiasis. Agglutination reaction. The results are shown in table 3c, in which the tendency was similar to precipitin reaction. However, as compared with the precipitin reaction, agglutination test seemed less reliable. VI. Further investigations on patients with candidiasis, related or unrelated diseases, and on healthy individuals As a result of the studies described above on the diagostic value of the Candida polysaccharide, it seemed to be useful in practical application. Thus, additional surveys were conducted to include other cases. The cases of superficial infections due to Candida sp. and pulmonary tuberculosis were examined. with particular interest because (a) in superficial infections there was a question as to whether the same results would be obtained as in the deep-seated infections, and (b) in pulmonary tuberculosis several investigators suggested that it might be more or less associated with candidiasis. Many clinicians of various hospitals were asked to test the antigenic solution on their patients. The report presented here is a survey of 232 individuals. From the results shown in tables 3a and 3b, the following concentrations of the antigen were considered to be diagostic standards suitable for excluding nonspecific reactions 0.1 cc of 1 Đg/cc of the antigen saline solution (the absolute content of inoculation, 0.1 Đg) in skin test, and 200 Đg cc solution in precipitin reaction. The technique and observation were similar to those of the above experiments. The individuals tested were grouped into four categories : Group I: candidiasis and suspected candidiasis, 19 cases Group II : pulmonary tuberculosis, 31 cases Group III: other diseases, 46 cases Group IV : healthy individuals, 136 cases All individuals tested were male and female adults. 1. Group I-cases of candidiasis and suspected candidiasis. As shown in table 4a, this group showed the highest percentage of positive reaction in both tests. It is interesting to note that cases of deep-seated infections (such as broncho-pulmonary candidiasis) showed a much more frequent positive reaction than those of superficial infections (such as cutaneous candidiasis). Oral candidiasis seemed to rank intermediately between the above two cases, and oral infections with ulcer also showed a more intense reaction when compared with superficial cases. The results of the skin tests were almost similar to those obtained by the precipitin reaction. The frequency of isolation of Candida sp. was positive in all cases, excepting one case of cutaneous candidiasis. 2. Group II-cases of pulmonary tuberculosis. Almost all of the patients

7 January 1957 SEROLOGIC DIAGNOSIS OF CANDIDIASIS 17 Table 4a. Skin Test and Precipitin Reaction Using Candida Polysaccharide Fraction and Isolation of Candida Species in 19 Cases of Candidiasis and Suspected Candidiasis Table 4b. Same Tests on 31 Cases of Pulmonary Tuberculosis Table 4c. Same Tests on 46 Cases with Various Diseases Other Than Candidiasis and Pulmonary Tuberculosis Table 4d. Skin Test and Precipitin Reaction Using Candida Polysaccharide Fraction and Agglutination Reaction hi 136 Healthy Individuals in this group were chronic cases under treatment in hospitals. Tuberculin skin test was done simultaneously. As shown in table 4b, the number of positive reaction of this group in the skin test and precipitin reaction was considerably

8 18 AKIBA, IWATA AND INOUYE Vol. 1, No. 1 higher than those of groups III and IV. The frequency of isolation of Candida sp. was also relatively high. 3. Group III- cases of diseases other than those of groups I and II. In this category were included a variety of infectious and non-infectious diseases, and the results are shown in table 4c. The positive reactions were conspicuously rare. 4. Group IV-cases of healthy individuals. This group consisted of all persons who appeared quite healthy. Nonspecific reactions were the rarest of all groups, as shown in table 4d. DISCUSSION Negroni(3) reported that in C. albicans, precipitin and complement fixation antigens are present in the polysaccharide and agglutinogens in the cell proper. He also stated that the active agent of substances sensitizing patients with candidiasis is polysaccharide in nature. Skinner" reported that the polysaccharide separated from cells of C. albicans gave a positive skin reaction in individuals who gave a positive reaction to oidiomycin. In this connection, the experiment presented in this paper also demonstrated that the polysaccharide fraction of C. albicans is an active agent in the skin test, both in animals and patients infected by Candida sp. The extraction procedure of Candida polysaccharide was initially performed according to Boivin's method, but the average yield was relatively small. Thus, the modification of Palmer and Gerlough's method was employed which gave a greater yield in comparison with Boivin's method. As a result of chemical studies, the characteristics of the polysaccharide fraction were that it consisted mainly of glucose and a small amount of other unqualified substances. Neither did it contain phosphorus nor show any specific absorption zone of ultraviolet ray. The degree of sensitivity of the skin to the polysaccharide fraction corresponded to the precipitin and agglutinin titers of sera on rabbits immunized with dead cells of Candida as well as the rabbits infected with living cells. The polysaccharide fraction prepared from C. albicans, strain 2, was quite similar in its antigenic activity to rabbits immunized or infected with different strains of C. albicans. On the contrary, a slight degree af cross reaction was observed on rabbits immunized with other Candida sp. The skin test and precipitin reaction, using the polysaccharide fraction, showed a considerably high specificity as far as the results obtained are concerned. Agglutination reaction seemed to be inferior in specificity and sensitivity as compared with the precipitin reaction using the polysaccharide. A suitable dose of antigen in the skin test on persons was determined so as not only to exclude non-specific reactions as much as possible, but also to permit the diagnosis of true candidiasis, especially in the case when the production of serum antibody is not so conspicuous. For the skin test, the result would be more significant if 0.1, 0.2 and 0.5 Đg of the antigen were simultaneously injected. For precipitin reaction, it is desirable to use at the same time,

9 January 1957 SEROLOGIC DIAGNOSIS OF CANDIDIASIS 19 the 500 Đg/cc solution besides the 200 Đg/cc solution of the antigen. In fact, it was confirmed that most of the doubtful positive reactions in the skin test with injections of 0.1 Đg or in the precipitin reaction with 200 Đg/cc solution turned definitely positive when a little more antigen was used. The skin reaction caused by the polysaccharide may be divided into two types, immediate and delayed. The majority of the reaction was the delayed type. Induration was not observed too frequently, and necrosis was never encountered with. The authors do not assert that the skin test or the precipitin reactin using the Candida polysaccharide can decide by themselves the diagnosis of candidiasis in every case, but expect that such tests may play an important role in diagnosing the disease as early and definitely as possible, if additional tests (mycological and clinical) are performed at the same time. Furthermore, the authors experienced that the antibody titer in patients could be correlated with the severity of the disease ; the titer dropping and the skin test turning negative as clinical improvement became noticeable in almost all cases, the data of which are not presented in this paper. SUMMARY The authors extracted the polysaccharide fraction from the celles of a strain of Candida albicans. This antigen was used in the diagnosis of candidiasis, especially the deep-seated infections, by skin test and precipitin reaction. The polysaccharide antigen proved to be considerably high in specificity and sensitivity from the results of animal experiments and clinical investigations on human cases. Therefore, the authors consider it a reliable antigen for the serologic diagnosis of candidiasis. The authors wish to express their sincere gratitude for the Grants in Aid for the Scientific Researches provided by the Ministry of Education, and also for the kind co-operation rendered by several doctors in various hospitals in carrying out the skin test, precipitin reaction and other tests. REFERENCES ( 1 ) Conant, N. F., Smith, D. T., Bakar, R. D., Callaway, J. L., and Martin, D. S., Manual of Clinical Mycology Saunders Co. (Philadelphia) p. 169, 2nd Edition, ( 2 ) Palmer, J.W. and Gerlough, T. D. L., Science 92, 155, ( 3 ) Negroni, P., Compt. Rend. Soc. Biol. 115, 342, ( 4 ) Skinner, C. E., Ktct. Rev. 11, 227, 1947.