Mycopathologia 102 :17-19 (1988) �9 Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands 17

Detection of circulating antibodies in patients affected by Chromoblastomycosis by Cladosporium carrionii using double immunodiffusion

Elena Villalba & Jos6 Francisco Yegres Area Ciencias de la Salud, Microbiologfa, Centro de Investigaciones Biomddicas, Universidad Nacional Experimental Francisco de Miranda, Coro, estado Falc6n, Venezuela

Accepted 12 December 1987

Key words: antibodies, Chromoblastomycosis, immunodiffusion, mycelial somatic antigens, culture filtrates antigens, serological test

Abstract

The antibodies in sera of patients affected by Chromoblastomycosis are detected using the technique of double immunodiffusion and the mycelial somatic antigens and the culture filtrates antigens of Cladosporium carri- onii. From the 13 sera tested 8 have given positive results. The fresh serum from a patient under treatment

gives 2 bands, while fresh serum from a non-treated patient gives 3 bands. The titre of antibodies was also determined for the two fresh sera, having found 1/4 for the patient under treatment and 1/32 for the non-treated one.

Introduction

It was in 1966, when the use of double immunodiffu-

sion was first reported to detect circulating antibod- ies in patients affected by Chromoblastomycosis caused by Fonsecaeapedrosoi. Somatic mycelial an-

tigens were used and the results were positive on 12 of the 13 serum tested [1].

After that date there has been no report of the use of this technique in Chromoblastomycosis cases, and this could occur since serological tests are of lit-

tle use in the diagnosis of this mycosis. However, it would be most convenient to make a follow up of the treatment. Currently new drugs are being used [2, 3, 4, 5]. Therapies are also rationalized, therefore it is very important to have an adequate technique to evaluate them.

The present paper deals with the results of the use and assessment of this technique in the detection of

circulating antibodies in patients affected by this mycosis caused by Cladosporium carrionii.

Materials and methods

Cladosporium carrionii was isolated in all patients studied [6]. One of these strains was used to obtain the antigens. Two types were used: culture filtrates (CF) and mycelial somatic antigens (MSA). The cul- tures were done in a YPG broth (glucose 1%, pep- tone 1%, levadure extract 1%) for the CF and in the

Sabouraud broth for the MSA. These were incubat- ed in repose for 4 weeks MSA and for different peri- ods of time CE

The CF were obtained by first passing cultures through a Whatman N ~ 2 paper and the filtered liq- uid was passed through a Millipore 0.45 mycras, then was concentrated by perevaporation at 4~ and

18

stored at - 2 0 ~ The mycelial was separated and washed with saline solution 0.85% (0.15 M). It was then resuspended in a small volume of this same so- lution and was macerated in a porcelain mortar. The

gross suspension was processed in the same manner as the culture filtrates.

To obtain serum, a blood sample by venous punc-

ture was taken. This serum was stored at - 2 0 ~ without preservatives. Before the test it was placed

at 56 ~ for 30 rain to inactivate complement. The immunodiffusion was performed according

to the Buckley and Murray method 1966 [1], but the

plates were incubated at 37 ~ during 12-48 hours, observed and incubation continued at room temper- ature for up to 10 days with daily observations.

Fig. 1. The results in a treated patient. The 3 antigens are culture filtrates with 50 days of incubation concentrated 20 • The arrows mark the band.

Results

After incubation, one or more bands were observed for the mycelial somatic antigens, concentrated 2 x , and the culture filtrates with 50-111 incubation

days, concentrated 2 0 - 30 x . The number of bands and the incubation period

in which they appear will depend on the serum stor- age period. It was found that fresh serum gave a greater number of bands and they appeared with only 18 - 20 hours of incubation at 37 ~ Apparent-

ly, the number of bands also varies with the evolu- tion and treatment of the illness. Mycelial somatic

antigens and culture filtrates give the same result. Sera from non-treated patients stored for 1 - 2 years

gave 1 - 2 bands but only if non-diluted sera were used. Fresh serum from a patient with 30 years evolu- tion, relapsed and under treatment, gave 2 bands (Fig. 1), while fresh serum from a non-treated pa- tient gave 3 bands (Fig. 2). From the 13 sera tested, 8 have given positive results. The negativity of the others might have been due to the loss of antibodies during storage. A positive serum from a patient with Paracoccidiodomycosis was tested and the results

were negative. The title of the antibodies was also determined for

the fresh sera having found 1/4 for the patient under treatment and 1/32 for the non treated one. Titles are the same for both types of antigens used.

Fig. 2. The results in a non-treated patient. The antigen is culture filtrates 54 days of incubation concentrated 20x.

Discussion

Results presented herein show the existence of precipitating antibodies in patients affected by Chromomycosis due to Cladosporiurn carrionii.

These antibodies may be detected using mycelial so- matic antigens or culture filtrates with more than 50

days incubation in repose. The results of the test will depend on the use of

fresh sera since storage for long periods causes losses or inactivity of the antibodies. It has been noted that this happens more frequently if the same sample is manipulated several times or is frozen and defrozen repeatedly and also if it is contaminated.

The number of bands and the title appear to be

indicative of the evolution of the illness and the an-

swer to the treatment. Unfortunately there are no previous results from the treated patient. A follow up, is being done on the other patient.

Acknowledgement

This work is supported in part by FUNDACITE F1.20.03.83.

References

i. Buckley HR, Murray IG: Precipitating antibodies in Chro-

momycosis. Sabouradia 1966; 5:78-80. 2. De Clercq D, Kakiesse M, De Vroey C, Mazebo P: Treatment

of Chromomycosis with a combination of 5 flurocytosine and

19

thiabendazole. Apropos of a Zairian case due to Fonsecaea pedrosoi. Ann Soc Belg Med Trop 1985; 65:95-97.

3. Deutsch PH, Quintiliani R: Ketoconazole. Conn Med 1984;

48:216-221. 4. Solano E, Hidalgo H, Castro A, Montero Gel F: Chromomy-

cosis treatment with a combination of thiabendazole and 5 fluorocytosine, 6 year follow up. Med Cutan ibero Lat Am 1983; 11:413-418.

5. Heyl T: Treatment of Chromomycosis with Itraconazole. Br J Dermatol 1985; 112:728-729.

6. Yegres F, Richard de Yegres N, Medina Ruiz E, Gonz~ilez Vivas R: Cromomicosis por Cladosporium carrionii en criadores de caprinos del Estado Falc6n. Investigaci6n Cl/nica 1985; 26:235 -246.

Address for offprints: Centro de Investigaciones Biom6dicas Calle E1 Tenis Area de Salud Mental Hospital General, Coro estado Falc6n Venezuela