Abstract
It is the author's purpose in this paper to help lead to a wider appreciation of the "immuno-allergy" SPOROTRICHIN test, by the study of two types of antigens in various concentrations. Following the preliminary investigation of LACAZ et a.l (1953), the author studied two antigens in greater detail: the first a suspension of the yeast-like elements of the Sporotrichum schencki; the second, an antigen rich in polysaccharides obtained from the same fungus, cultured at 37° with the FRANCIS and SABOURAUD media, extracted by autoc1aving a suspension of Sporotrichum schencki, yeast like type according to the technique of NORDÉN (1951) modified by FAVA NETTO (1955) for the extraction of the polysaccharides of Paracoccidioides brasiliensis. In the preface and introduction, reasons are given for the belief that the matter is an important one in both its theoretical and practical aspects. From the practical point of view it is necessary to know the exact validity of the Sporotrichin test as a diagnostic method, From the theoretical view point it is hoped to lead to a better understanding of the immuno-allergy of this mycosis, especially that aspect dealing with the possible existence of an "sporotrichosis infection" which, once proved, would put the Sporotrichin test in the same category as the other intradermal tests used for certain deep mycosis such as histoplasmosis and coccidioidomycosis. After reviewing the literature of the subject it was felt that further studies were required to lead to a better evaluation of this reaction. In the second chapter the antigen at present in use are investigated. That obtained from the suspension of Sporotrichum schencki in its yeast like phase was standardized by spectroscopy in 3 different concentrations; the polysaccharide antigen was extracted from the original substances with acetone and ether and later by autoclaving at 120°. The extract thus obtained was diluted to 1/100, 1/500 and 1/1000 and used as the antigen in the reaction, Tests were not made using filtered Sporotrichin because of the difficulty of standardizing such an antigen. The technique of the test is described, it is that normally used for skin tests, and also the criteria employed in reading the reaction. These were those proposed by the Department of Health, Education and Welfare of Public Health Service (1954) for histoplasmosis. Positive results were divided into groups one, two, three, or four to give the reader an idea of the degree of positivity obtained. The method used to study the histopathology of the reaction is described. Both types of antigen (N 2 and P 1) are injected into patients with sporotrichosis and biopsies performed, under local anesthesic with ethyl chloride at different lengths of time to give an idea of the sequence of events taking place in the histopathology. The skin biopsies were fixed in 10% formalin embedded in paraffin and sectioned 5 to 6 micra thick, using hematoxylin and eosin as stain. When it was thought advisable, sections were made and stained by other methods, periodic acid-Schiff and cresyl violet. Table IV shows the distribution of patients, in whom biopsies were made, of the reaction to antigen N 2 to the time between the injection of the antigen and the removal of the skin fragment; in Table V the same data are given for antigen P 1. Chapter III sets out the results obtained in various patients which were distributed in the following manner:
(a) Paciente with sporotrichosis (both active and past cases). These consisted of five groups, i. e.:
1st. group (18 cases) patients with active sporotrichosis who were tested with sporotrichin N 1, N 2 and N 3. A positive reaction was obtained with all of these (Table VI). A greater number of strongly positive reactions was obtained with antigens N 1 than N 2 and N 3, the result which would be expected. In a similar manner there were a greater number of strong positive with N 2 than N 3.
2nd. group (21 cases) patients with active sporotrichosis who were tested with sporotrichin N 2 and N 3. As in the former group, the test was positive in every case receiving antigen N 2; one case receiving N 3 was negative.
3rd. group (26 cases) patients with active sporotrichosis who were tested with sporotrichin N 2. A positive reaction was given by 25 of the 26 cases tested. Taking the three groups together it can be seen that of those individual with active sporotrichosis who were given the sporotrichin test, using antigen N 2, 64 out of 65 gave a positive result (98.46%).
4th. group (23 cases) patients with active sporotrichosis who as well as being tested with sporotrichin N 2 were also tested with the polysaccharide antigen obtained from cultures of Sporotrichum schencki by the above mentioned method. This later antigen was diluted to 1/100 (P 1), 1/500 (P 2) and 1/1000 (P 3) and injected intradermally. With antigen P 1, 21 positive results were obtained (91. 30%) and 2 negative results: with P 2, 19 positives (82.6 %) and 4 negatives ; with P 3, 13 negatives and 10 positives (43.47%).
5th. group (5 cases) patients in whom the sporotrichin test was made after a clinical cure of the disease. The time elapsed since the cure varied between 3 months and 7 years. The test was positive in all 5 cases with antigens N 2, P 1 and P. 2. With antigen P 3 a positive test was obtained in 3 cases and a negative in 2.
(b) Patients with other mycosis
Twenty five patients were studied, 23 with active disease and 2 cured cases. ln all of these the test was performed with antigens N 1 and N 2 and in 22 cases with P 1, P 2 and P 3 in addition. A positive result was obtained with the N antigens in one case of Tinea corporis (Nr. 23), in one of blastomycosis with N 1 (but negative with N 2); a positive with both N 1 and N 2 was given by 2 cases of blastomycosis and 2 of Tinea pedis with "id" (Nrs, 82, 87), (N 1 - 24% and N 2 - 16 positives in this group). With antigen P the following positive results were obtained: one case reacted positively to P 1 (Nr. 79) another positively to P 1 and P 2 (Nr. 87) and a third to P 1, P 2 and P 3 (Nr. 82). (P 1 - 13.63%, P 2 - 9.09% and P 3 - 4.54% positives in this group.)
(c) Miscellaneous cases
Fifty eight other cases were studied with various diseases together with several normal individuals. The test was done in every case with antigens N 1 and N 2 and in 54 of the 58 cases with the polysaccharide antigen, in the three before mentioned dilutions, as well. The result with N 1 was negative in 49 cases, and positive in 9 (15.51 %); with N 2, positive in 5 (8.62 %) and negative in 53. With the P 1 antigen, 5 positive reactions (9.25 %) were obtained in the 54 cases tested; with P 2, 4 positives (7.40 %) and 50 negatives and with P 3, 2 positives (3.70%) and 52 negatives. In the same chapter, section D, a summary is given of the macroscopic changes of the reaction. The local changes observed between the 48th and 72nd hours were: erythema, papular eruptions, papule with edema, tubercles, nodules, vesicles, pustules and ulceration. The basic lesion most frequently seen was edematous papule. Vesicles, pustules and ulceration were found as early as 48 - 72 hours in those lesions which were strongly positive. The author's findings with regard to the late changes with sporotrichin confirmed those of MIRANDA & colab. (1948). Generalized reactions were rarely seen and when present were not of marked intensity. In section E of chapter III the histopathology of the lesion is described. An overall appreciation of the biopsies taken shows that the histology with sporotrichin passes from the phase of transient, local proliferation and later demonstrates the picture of exsudation. This new productive reaction is followed by the appearance of granulation tissue. The reaction to antigen N 2 is more marked than that to P l. From these results it can be concluded that, of those antigens tested, the most satisfactories are N 2 and P 1. Antigen N 2 shows itself to be extremely sensitive (positive reactions being obtained in 64 cases out of 65 in which both antigens were used). With regard to the specificity; in that group of patients with other mycosis, with N 1, 6 positive reactions were obtained (24 %) and with N 2, 4 (16 %) and in the group of miscellaneous cases, with N 1, 9 positive results were seen (16.07 %) and with N 2, 5 (9.10 %). In chapter IV coments are made upon the results obtained. It is shown that the use of sporotrichin in greater dilution (N 2) than was previously used by LACAZ & colab, (1953), gives equivalent results in respect to the sensitivity of the reaction and better results with regard to the specificity. However even with this dilution there occurred some positive reactions, probably group reactions, in patients with other mycosis. Some positive results obtained with patients who had neither active nor past sporotrichosis, nor other active mycosis, suggest the existence of "sporotrichosis infections" as MACKINNON & colab. (1953) have suggested. A comparative study between the antigen prepared from the yeast like phase of Sporotrichuni schencki (N 2) and the polysaccharide extract of the same mould shows that there is no advantage in using the latter antigen. Its sensibility was less and there also occurred, though less in number, positive reactions in other patients without sporotrichin. From this study it can be seen that the Sporotrichin test, critically interpretated together with the clinical findings, can constitute a valuable armament in the early presumptive diagnosis of sporotrichosis. The test should be carried out together with the making of cultures. But it should be realized that this indirect test is not more important than the isolation of the fungus from the lesions. No conclusions can be reached, from the material studied, as to the time at which the test becomes positive nor was it possible to establish a clear relationship of the clinical manifestation or duration of the disease with the degree of positive reaction obtained. Histological studies showed a greater tissue reaction to an antigen containing cellular elements to that of one entirely in solution. The histological reaction of sporotrichin shows a certain similarity to that of sporotrichosis which suggests that there exists in this latter an allergic component, which would explain the difficulty in finding, microscopically, the parasite in the lesions.
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