Mycopathologia 118: 127-131, 1992. 9 1992 KluwerAcademic Publishers. Printedin the Netherlands.
Immunodiffusion test for diagnosing basidiobolomycosis P. Imwidthaya I & S. Srimuang 2 i Department of Microbiology, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand; 2Division of Infectious Disease and Host Defence Unit, Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand Received 3 August I990; accepted in revised form 13 November 1991
Key words: Basidiobolomycosis, immunodiffusion
Abstract
An immunodiffusion test was developed for the diagnosis of basidiobolomycosis. When culture filtrate antigen (CFA) from Basidiobolus ranarum was reacted against two human patient and two rabbit antisera, 2 precipitin bands, inner (N) and outer (Y), were revealed for both patient and rabbit antisera. A line of identity was also observed between precipitin bands obtained with patient and rabbit sera. When CFA from B. ranarum (B CFA) was reacted against rabbit sera which contained antibody to Conidiobolus coronatus and Pythium insidiosum, 1 precipitin band corresponding to inner band (N) was observed. This finding showed that B. ranarum, C. coronatus and P. insidiosum shared at least one common antigen. After B CFA was absorbed with Pythium rabbit antiserum, the inner precipitin line that occurred between B CFA and rabbit antisera of Pythium and Conidiobolus disappeared. However, with Basidiobolus rabbit antiserum, the result did not change. The antigens which could be demonstrated by inner (N) and outer (Y) precipitin bands were heat stable at 56 ~ for 30 min. The titer of the antibodies specific to these antigens decreased as the lesions subsided. When B. ranarum CFA was reacted against sera from 20 apparently normal persons, 20 diabetes mellitus patients, 5 aspergillosis patients, 2 candidosis patients and 3 pythiosis patients, no precipitin band was found. B. ranarum CFA was also treated with each rabbit antiserum specific to Candida albicans, Malassezia furfur and Aspergillus fumigatus. No precipitin bands occurred with any of these antisera. Thus, this test was found to be practical, sensitive and specific, and can be used to monitor patients infected with Basidiobolus ranarum.
Introduction
Basidiobolomycosis was first reported in 1956 from Indonesia [1]. This disease has occurred sporadically in Thailand [2-4], and the organism causing it can be isolated from toads excreta [5].
The causative agent is placed in the class Zygomycetes, order Entomophthorales. Basidiobolus ranarum, the organism causing basidiobolomycosis, contaminates excreta of reptiles and amphibians, and humans can be infected. The lesions of subcutaneous basidiobolomycosis
128 commonly develop on the lower extremities and exposed areas. Definite diagnosis is based on isolation of the causative organism from lesions. In November 1989, a 3-year old boy from Angthong Province, central Thailand, was admitted to Siriraj Hospital, Mahidol University, with subcutaneous zygomycosis. His parents worked in sugar cane fields and had carried the boy to the fields since he was 6 months of age. About 7 months before admission he developed subcutaneous lesions which progressively enlarged. On admission, the boy looked undernourished with temperture 39 ~ pulse 150/rain and respiration 32/rain. Subcutaneous tissue masses of firm consistency were present on the trunk, chest and extremities. The lesions varied in size from 10• to 1 • and the covering skin looked slightly inflammed. Blood examination revealed hematocrit 27%, white count 33,700, polymorphs 50%, lymphocytes 18%, eosinophils 29% and basophils 3%. His cell-mediated immune response was within normal limits. Because the patient had high fever, the biopsy schedule was postponed for 2 weeks after admission. Culture from the biopsy specimen revealed that the organism could grow well on Sabouraud dextrose agar at room temperature and at 37 ~ The colonies with waxy surface appeared within 3 days. Microscopic examination revealed non-septate hyphae with smooth walled zygospores, globose and adhesive conidia. Based on Rippon's description of B. ranarum (1982) the organism was identified as B. ranarum [6]: An additional case came from Khon Kaen Province, Northeast Thailand. The patient was 38-year old male farmer, who had developed a subcutaneous lesion on his right leg. A skin biopsy from the lesion revealed non-septate hyphae which the microbiologist in Kohn Kaen identified as B. ranarum. The serum was sent for an immunodiffusion test. The two patients were treated with co-trimoxazole. This immunodiffusion test was developed as an aid in diagnosing basidiobolomycosis. It could be particularly helpful in cases where the biopsy schedule has to be postponed, and it might be useful in monitoring the disease.
Materials and methods
Culture. B. ranarum, designated B, was obtained from subcutaneous lesions found in the 3-year old boy from Angthong Province. Sera. Sera were obtianed from the 3-year old boy who had subcutaneous basidiobolomycosis, at 0, 3, and 6 weeks after treatment, designated B0, B3 and B6, respectively. The serum obtained from the Khon Kaen subject with subcutaneous basidiobolomycosis was designated B~. For control purposes, the following human sera were also tested: 20 from apparently healthy persons, 20 from diabetes mellitus patients, 3 from proven pythiosis, 2 from proven candidosis and 5 from proven aspergillosis cases. Each rabbit serum which contained antibody to Candida albicans, Malassezia furfur, Conidiobolus coronatus or Aspergillus fumigatus was also used for testing cross reaction with B. ranarum antigen. Antigen preparation. A culture of B. ranarum was transferred to brain-heart infusion agar (Difco Laboratories, Detroit, Mich.) and incubated at 37~ for 7 days. Small portions of growth from these cultures were transferred to each of two 1-1itre flasks containig 500ml of brain-heart infusion broth. The flasks were incubated at 37 ~ on a rotating shaker at 150 rpm for 10 days. The cultures were killed with merthiolate (0.02%), filtered and concentrated 20-fold through dialysis membrane using negative pressure (700mmHg). Protein concentration of B was 500mg%. This preparation was designated as culture filtrate antigen (CFA). Absorption of culture filtrate antigen. To eliminate non-specific precipitation bands, Pythium rabbit antiserum (1 ml) was placed in a glass tube and dried by bubbling air through the tube. Culture filtrate antigen (1 ml) was added to the dried antiserum. The antigen-antiserum mixture was agitated to dissolve the antigen completely and incubated at 37 ~ for 2 hr in a water bath with an agitator. After incubation, the reactants were centrifuged for 45 min at 12,063 • g. The super-
129 natant was decanted and tested by immunodiffusion for evidence of absorption [7].
Rabbit reference B. ranarum ant&era production. Two New Zealand white rabbits were initially bled, and their serum was run in a macroimmunodiffusion plate against B CFA to reveal any antibody present before immunization. Two rabbits were injected subcutaneously on day 1 with 0.5 ml of B CFA in 0.5 ml of Freund's complete adjuvant. During week 3, the rabbits were injected with 0.5 ml of B CFA in 0.5 ml of Freund's incomplete adjuvant. Bleeding and immunization were alternately performed weekly until a significant precipitin line was revealed in the immunodiffusion plate (8 weeks). ID test. Agar gel double immunodiffusion was performed in 100-mm-diameter plastic petri dishes. Each petri dish was filled with 7.5 ml of purified 1% phenolized agar. Wells 3 mm in diameter, 3 mm apart, were punched out in a hexagonal pattern. H u m a n basidiobolomycosis sera and other sera containing antibodies to various fungi were placed in the wells and allowed to diffuse for i hr at room temperature before the reference antigen was added. They were then incubated for 48 hr in a humidity chamber at room temperature. Dilution of patients sera was performed with sterile normal saline solution.
Results
ID studies were carried out with 10-day old B CFA and rabbit reference B antisera. Five precipitin bands were revealed with both rabbit antisera. When B CFA was tested with sera obtained from two cases of subcutaneous basidiobolomycosis patients, precipitin bands identical to those of rabbit reference B antisera could be observed (Fig. 1). The serum from the 3-year old boy decreased in titer from 1:32 to 1:2 during the period of 6 weeks after treatment (Table 1). The serum from the 38-year old man showed an antibody titer of 1 : 4. One line of partial identity to the inner line,
Fig. 1. Reactivity of B. ranarum culture filtrate antigen (B) with rabbit Basidiobolus antiserum (Bo), rabbit ConidioboIus antiserum (C), rabbit Pythium antiserum (P) and human subcutaneous basidiobolomycosis (Bh). Two lines of identity were observed between precipitin lines obtained with rabbit and human subcutaneous basidiobolomycosis. One line corresponding to the inner line was observed with rabbit Conidiobolus antiserum and rabbit Pythium antiserum.
Table I. Immunodiffusion reactions of human sera from subcutaneous basidiobolomycosis Disease state
Week of blood collection (after treatment with Co-trimoxazole)
Titer of sera reacted with reference culture filtrate antigen
Subcutaneous basidiobolomycosis
0 3 6
1 : 32 1:8 1:2
obtained with rabbit reference B antisera, was revealed with rabbit sera which contain antibody to Pythium insidiosurn. A line of partial identity was also observed between rabbit anti-Conidiobolus coronatus and rabbit anti-Pythium ins# diosum. After B CFA was absorbed with Pythium rabbit antiserum, the inner precipitin line that occurred between B CFA and rabbit antisera against Pythium and Conidiobolus disappeared. In contrast, the results did not change with the
130 Table 2. Immunodiffusion reactions of sera obtained from patients with subcutaneous phycomycosis and human control sera with B culture filtrate antigen Disease state
Subcutaneous basidiobolomycosis Pythiosis Candidosis Aspergillosis Diabetes mellitus Normal
No. of human sera tested
No. of positive results
No. of precipitin lines identical to those of rabbit anti-B CFA antisera
2
2
2
3 2 5 20 20
0 0 0 0 0
0 0 0 0 0
Table 3. Immunodiffusion reactions of rabbit antisera with B culture filtrate antigen
Fig. 2. Reactivity of B. ranarum culture filtrate antigen (B) with rabbit Basidiobolus antiserum (Bo), rabbit Conidiobolus antiserum (C) and rabbit Pythium antiserum (P). Rabbit Basidiobolus antiserum showed inner and outer precipitin lines. Rabbit antiserum specific to Pythium showed partial identical inner lines with rabbit Basidiobolus antiserum and antiserum to Conidiobolus. After B CFA was absorbed with rabbit Pythium antiserum (AB), the inner precipitin line with P and C disappeared but 2 precipitin lines were still observed with Bo.
use of Basidiobolus antiserum and B CFA which was absorbed with Pythium rabbit antiserum. Other sera showed no precipitin line with B CFA (Fig. 2; Tables 2 and 3).
Discussion
When CFA from B. ranarum was reacted against sera from basidiobolomycosis patients and rabbit Basidiobolus antisera, 2 precipitin bands, an inner and outer, were revealed with both patient and rabbit antisera and they also showed a pattern of identity. The antigens forming these two precipitation lines were heat stable at 56 ~ for 30 rain. This finding corresponds with that reported by Yangco et al. [8], who named the inner line as N and the outer line as Y.
Antibody to
No. of No. of precipitin rabbit tested lines identical to those of rabbit anti-B CFA antisera
Candida albicans Malassezia furfur Aspergillus fumigatus
I 1 1
0 0 0
In Thailand, Pythium insidiosum can cause subcutaneous infection as well as Basidiobolus [9] and sometimes the lesions look alike. With the ID test, pythiosis antiserum showed lines of partial identity with Basidiobolus antiserum. However, after the absorption of B. ranarurn antigen with antiserum specific to P. insidiosurn, this line of partial identity disappeared while the precipitation lines were still observed between the absorbed antigen and antiserum to Basidiobolus. Kaufman et al. [10] worked on immunodiffusion test for serodiagnosing subcutaneous zygomycosis found that homologous rabbit antisera B. ranarum and C. coronatus were each found to have five specific antigens and results of tests with heterologous antisera indicated that all of the species shared at least one antigen. Moreover, Kaufman et al., performed ID test with sera from human and horse with proven basidiobolomycosis and conidiobolomycosis. Thus, from Kaufman et al.,
131 and our work confirmed that, the ID test appears to be useful for the diagnosis of basidiobolomycosis. The titer of antibodies to B. ranarum decreased after the lesion had subsided. Our work also confirms that of Yangco et al. in that Basidiobolus and ConidioboIus were found to share c o m m o n antigens and, m o r e o v e r , we discovered that B. ranarum also shared at least one c o m m o n antigen with P. insidiosum which could be identified by a line of partial identity with the inner precipitation line which occurred between B. ranarum and its homologous antiserum.
Acknowledgements The authors wish to thank Dr. Warren Brockelman and Dr. Napatawn Banchuin for correcting the manuscript, and Dr. Suttipan Sarasombat for concentrating the antigen.
References 1. Joe LK, Njo-Tioei E, Pohon A, Van der Meulen A, Emmons CW. Basidiobolus ranarum as a cause of subcutaneous mycoses in Indonesia. Arch Dermatol 1956; 74: 378-83. 2. Chantarakul N, Talalak P, Vareenil J. Phycomycosis. J. med Ass Thailand 1965; 48: 740-9. 3. Thasnakorn P, Sribooma V, Chantarakul N, Bhadrakom S. Subcutaneous mycosis due to Basidiobolus meristosporus: report of a case. J. Med Ass Thailand 1969; 52: 372-7. 4. Thianprasit M. Subcutaneous phycomycosis in Thailand. 5th Int Congr of Infectious Diseases (Vienna). Separatum 1970; 43-45-A 11/3-5. 5. Imwidthaya S, Plangpatanapanichya A. Sripathomswat N. Basidiobolus in Toad's excreta in Thailand. J. Med Ass Thailand 1982; 65: 420-5. 6. Rippon JW. Medical mycology, 2nd ed. Philadelphia: WB Saunders Comp 1982: 303-14. 7. Jones KW, Kaufman L. Development and evaluation of an immunodiffusion test for diagnosis of systemic zygomycosis (mucormycosis): preliminary report. J Clin Microbiol 1978; 7: 97-101. 8. Yangco BG, Nettlow A, Okafor JI, Park J, Te-Strake D. Comparative antigenic studies of species of Basidiobolus and other medically important fungi. J Clin Microbiol 1986; 34: 679-82. 9. Thianprasit M. Cutaneous Pythiosis in Human-Being. Abstract in Siriraj 100th Academic Meeting, Mahidol University. Bangkok, Thailand, 18-22 April 1988: 94. i0. Kaufman L, Mendoza L, Standard PG. Immunodiffusion test for serodiagnosing subcutaneous zygomycosis. J Clin Microbiol 1990; 28: 1887-1890.
Immunodiffusion test for diagnosing basidiobolomycosis P. Imwidthaya I & S. Srimuang 2 i Department of Microbiology, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand; 2Division of Infectious Disease and Host Defence Unit, Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand Received 3 August I990; accepted in revised form 13 November 1991
Key words: Basidiobolomycosis, immunodiffusion
Abstract
An immunodiffusion test was developed for the diagnosis of basidiobolomycosis. When culture filtrate antigen (CFA) from Basidiobolus ranarum was reacted against two human patient and two rabbit antisera, 2 precipitin bands, inner (N) and outer (Y), were revealed for both patient and rabbit antisera. A line of identity was also observed between precipitin bands obtained with patient and rabbit sera. When CFA from B. ranarum (B CFA) was reacted against rabbit sera which contained antibody to Conidiobolus coronatus and Pythium insidiosum, 1 precipitin band corresponding to inner band (N) was observed. This finding showed that B. ranarum, C. coronatus and P. insidiosum shared at least one common antigen. After B CFA was absorbed with Pythium rabbit antiserum, the inner precipitin line that occurred between B CFA and rabbit antisera of Pythium and Conidiobolus disappeared. However, with Basidiobolus rabbit antiserum, the result did not change. The antigens which could be demonstrated by inner (N) and outer (Y) precipitin bands were heat stable at 56 ~ for 30 min. The titer of the antibodies specific to these antigens decreased as the lesions subsided. When B. ranarum CFA was reacted against sera from 20 apparently normal persons, 20 diabetes mellitus patients, 5 aspergillosis patients, 2 candidosis patients and 3 pythiosis patients, no precipitin band was found. B. ranarum CFA was also treated with each rabbit antiserum specific to Candida albicans, Malassezia furfur and Aspergillus fumigatus. No precipitin bands occurred with any of these antisera. Thus, this test was found to be practical, sensitive and specific, and can be used to monitor patients infected with Basidiobolus ranarum.
Introduction
Basidiobolomycosis was first reported in 1956 from Indonesia [1]. This disease has occurred sporadically in Thailand [2-4], and the organism causing it can be isolated from toads excreta [5].
The causative agent is placed in the class Zygomycetes, order Entomophthorales. Basidiobolus ranarum, the organism causing basidiobolomycosis, contaminates excreta of reptiles and amphibians, and humans can be infected. The lesions of subcutaneous basidiobolomycosis
128 commonly develop on the lower extremities and exposed areas. Definite diagnosis is based on isolation of the causative organism from lesions. In November 1989, a 3-year old boy from Angthong Province, central Thailand, was admitted to Siriraj Hospital, Mahidol University, with subcutaneous zygomycosis. His parents worked in sugar cane fields and had carried the boy to the fields since he was 6 months of age. About 7 months before admission he developed subcutaneous lesions which progressively enlarged. On admission, the boy looked undernourished with temperture 39 ~ pulse 150/rain and respiration 32/rain. Subcutaneous tissue masses of firm consistency were present on the trunk, chest and extremities. The lesions varied in size from 10• to 1 • and the covering skin looked slightly inflammed. Blood examination revealed hematocrit 27%, white count 33,700, polymorphs 50%, lymphocytes 18%, eosinophils 29% and basophils 3%. His cell-mediated immune response was within normal limits. Because the patient had high fever, the biopsy schedule was postponed for 2 weeks after admission. Culture from the biopsy specimen revealed that the organism could grow well on Sabouraud dextrose agar at room temperature and at 37 ~ The colonies with waxy surface appeared within 3 days. Microscopic examination revealed non-septate hyphae with smooth walled zygospores, globose and adhesive conidia. Based on Rippon's description of B. ranarum (1982) the organism was identified as B. ranarum [6]: An additional case came from Khon Kaen Province, Northeast Thailand. The patient was 38-year old male farmer, who had developed a subcutaneous lesion on his right leg. A skin biopsy from the lesion revealed non-septate hyphae which the microbiologist in Kohn Kaen identified as B. ranarum. The serum was sent for an immunodiffusion test. The two patients were treated with co-trimoxazole. This immunodiffusion test was developed as an aid in diagnosing basidiobolomycosis. It could be particularly helpful in cases where the biopsy schedule has to be postponed, and it might be useful in monitoring the disease.
Materials and methods
Culture. B. ranarum, designated B, was obtained from subcutaneous lesions found in the 3-year old boy from Angthong Province. Sera. Sera were obtianed from the 3-year old boy who had subcutaneous basidiobolomycosis, at 0, 3, and 6 weeks after treatment, designated B0, B3 and B6, respectively. The serum obtained from the Khon Kaen subject with subcutaneous basidiobolomycosis was designated B~. For control purposes, the following human sera were also tested: 20 from apparently healthy persons, 20 from diabetes mellitus patients, 3 from proven pythiosis, 2 from proven candidosis and 5 from proven aspergillosis cases. Each rabbit serum which contained antibody to Candida albicans, Malassezia furfur, Conidiobolus coronatus or Aspergillus fumigatus was also used for testing cross reaction with B. ranarum antigen. Antigen preparation. A culture of B. ranarum was transferred to brain-heart infusion agar (Difco Laboratories, Detroit, Mich.) and incubated at 37~ for 7 days. Small portions of growth from these cultures were transferred to each of two 1-1itre flasks containig 500ml of brain-heart infusion broth. The flasks were incubated at 37 ~ on a rotating shaker at 150 rpm for 10 days. The cultures were killed with merthiolate (0.02%), filtered and concentrated 20-fold through dialysis membrane using negative pressure (700mmHg). Protein concentration of B was 500mg%. This preparation was designated as culture filtrate antigen (CFA). Absorption of culture filtrate antigen. To eliminate non-specific precipitation bands, Pythium rabbit antiserum (1 ml) was placed in a glass tube and dried by bubbling air through the tube. Culture filtrate antigen (1 ml) was added to the dried antiserum. The antigen-antiserum mixture was agitated to dissolve the antigen completely and incubated at 37 ~ for 2 hr in a water bath with an agitator. After incubation, the reactants were centrifuged for 45 min at 12,063 • g. The super-
129 natant was decanted and tested by immunodiffusion for evidence of absorption [7].
Rabbit reference B. ranarum ant&era production. Two New Zealand white rabbits were initially bled, and their serum was run in a macroimmunodiffusion plate against B CFA to reveal any antibody present before immunization. Two rabbits were injected subcutaneously on day 1 with 0.5 ml of B CFA in 0.5 ml of Freund's complete adjuvant. During week 3, the rabbits were injected with 0.5 ml of B CFA in 0.5 ml of Freund's incomplete adjuvant. Bleeding and immunization were alternately performed weekly until a significant precipitin line was revealed in the immunodiffusion plate (8 weeks). ID test. Agar gel double immunodiffusion was performed in 100-mm-diameter plastic petri dishes. Each petri dish was filled with 7.5 ml of purified 1% phenolized agar. Wells 3 mm in diameter, 3 mm apart, were punched out in a hexagonal pattern. H u m a n basidiobolomycosis sera and other sera containing antibodies to various fungi were placed in the wells and allowed to diffuse for i hr at room temperature before the reference antigen was added. They were then incubated for 48 hr in a humidity chamber at room temperature. Dilution of patients sera was performed with sterile normal saline solution.
Results
ID studies were carried out with 10-day old B CFA and rabbit reference B antisera. Five precipitin bands were revealed with both rabbit antisera. When B CFA was tested with sera obtained from two cases of subcutaneous basidiobolomycosis patients, precipitin bands identical to those of rabbit reference B antisera could be observed (Fig. 1). The serum from the 3-year old boy decreased in titer from 1:32 to 1:2 during the period of 6 weeks after treatment (Table 1). The serum from the 38-year old man showed an antibody titer of 1 : 4. One line of partial identity to the inner line,
Fig. 1. Reactivity of B. ranarum culture filtrate antigen (B) with rabbit Basidiobolus antiserum (Bo), rabbit ConidioboIus antiserum (C), rabbit Pythium antiserum (P) and human subcutaneous basidiobolomycosis (Bh). Two lines of identity were observed between precipitin lines obtained with rabbit and human subcutaneous basidiobolomycosis. One line corresponding to the inner line was observed with rabbit Conidiobolus antiserum and rabbit Pythium antiserum.
Table I. Immunodiffusion reactions of human sera from subcutaneous basidiobolomycosis Disease state
Week of blood collection (after treatment with Co-trimoxazole)
Titer of sera reacted with reference culture filtrate antigen
Subcutaneous basidiobolomycosis
0 3 6
1 : 32 1:8 1:2
obtained with rabbit reference B antisera, was revealed with rabbit sera which contain antibody to Pythium insidiosurn. A line of partial identity was also observed between rabbit anti-Conidiobolus coronatus and rabbit anti-Pythium ins# diosum. After B CFA was absorbed with Pythium rabbit antiserum, the inner precipitin line that occurred between B CFA and rabbit antisera against Pythium and Conidiobolus disappeared. In contrast, the results did not change with the
130 Table 2. Immunodiffusion reactions of sera obtained from patients with subcutaneous phycomycosis and human control sera with B culture filtrate antigen Disease state
Subcutaneous basidiobolomycosis Pythiosis Candidosis Aspergillosis Diabetes mellitus Normal
No. of human sera tested
No. of positive results
No. of precipitin lines identical to those of rabbit anti-B CFA antisera
2
2
2
3 2 5 20 20
0 0 0 0 0
0 0 0 0 0
Table 3. Immunodiffusion reactions of rabbit antisera with B culture filtrate antigen
Fig. 2. Reactivity of B. ranarum culture filtrate antigen (B) with rabbit Basidiobolus antiserum (Bo), rabbit Conidiobolus antiserum (C) and rabbit Pythium antiserum (P). Rabbit Basidiobolus antiserum showed inner and outer precipitin lines. Rabbit antiserum specific to Pythium showed partial identical inner lines with rabbit Basidiobolus antiserum and antiserum to Conidiobolus. After B CFA was absorbed with rabbit Pythium antiserum (AB), the inner precipitin line with P and C disappeared but 2 precipitin lines were still observed with Bo.
use of Basidiobolus antiserum and B CFA which was absorbed with Pythium rabbit antiserum. Other sera showed no precipitin line with B CFA (Fig. 2; Tables 2 and 3).
Discussion
When CFA from B. ranarum was reacted against sera from basidiobolomycosis patients and rabbit Basidiobolus antisera, 2 precipitin bands, an inner and outer, were revealed with both patient and rabbit antisera and they also showed a pattern of identity. The antigens forming these two precipitation lines were heat stable at 56 ~ for 30 rain. This finding corresponds with that reported by Yangco et al. [8], who named the inner line as N and the outer line as Y.
Antibody to
No. of No. of precipitin rabbit tested lines identical to those of rabbit anti-B CFA antisera
Candida albicans Malassezia furfur Aspergillus fumigatus
I 1 1
0 0 0
In Thailand, Pythium insidiosum can cause subcutaneous infection as well as Basidiobolus [9] and sometimes the lesions look alike. With the ID test, pythiosis antiserum showed lines of partial identity with Basidiobolus antiserum. However, after the absorption of B. ranarurn antigen with antiserum specific to P. insidiosurn, this line of partial identity disappeared while the precipitation lines were still observed between the absorbed antigen and antiserum to Basidiobolus. Kaufman et al. [10] worked on immunodiffusion test for serodiagnosing subcutaneous zygomycosis found that homologous rabbit antisera B. ranarum and C. coronatus were each found to have five specific antigens and results of tests with heterologous antisera indicated that all of the species shared at least one antigen. Moreover, Kaufman et al., performed ID test with sera from human and horse with proven basidiobolomycosis and conidiobolomycosis. Thus, from Kaufman et al.,
131 and our work confirmed that, the ID test appears to be useful for the diagnosis of basidiobolomycosis. The titer of antibodies to B. ranarum decreased after the lesion had subsided. Our work also confirms that of Yangco et al. in that Basidiobolus and ConidioboIus were found to share c o m m o n antigens and, m o r e o v e r , we discovered that B. ranarum also shared at least one c o m m o n antigen with P. insidiosum which could be identified by a line of partial identity with the inner precipitation line which occurred between B. ranarum and its homologous antiserum.
Acknowledgements The authors wish to thank Dr. Warren Brockelman and Dr. Napatawn Banchuin for correcting the manuscript, and Dr. Suttipan Sarasombat for concentrating the antigen.
References 1. Joe LK, Njo-Tioei E, Pohon A, Van der Meulen A, Emmons CW. Basidiobolus ranarum as a cause of subcutaneous mycoses in Indonesia. Arch Dermatol 1956; 74: 378-83. 2. Chantarakul N, Talalak P, Vareenil J. Phycomycosis. J. med Ass Thailand 1965; 48: 740-9. 3. Thasnakorn P, Sribooma V, Chantarakul N, Bhadrakom S. Subcutaneous mycosis due to Basidiobolus meristosporus: report of a case. J. Med Ass Thailand 1969; 52: 372-7. 4. Thianprasit M. Subcutaneous phycomycosis in Thailand. 5th Int Congr of Infectious Diseases (Vienna). Separatum 1970; 43-45-A 11/3-5. 5. Imwidthaya S, Plangpatanapanichya A. Sripathomswat N. Basidiobolus in Toad's excreta in Thailand. J. Med Ass Thailand 1982; 65: 420-5. 6. Rippon JW. Medical mycology, 2nd ed. Philadelphia: WB Saunders Comp 1982: 303-14. 7. Jones KW, Kaufman L. Development and evaluation of an immunodiffusion test for diagnosis of systemic zygomycosis (mucormycosis): preliminary report. J Clin Microbiol 1978; 7: 97-101. 8. Yangco BG, Nettlow A, Okafor JI, Park J, Te-Strake D. Comparative antigenic studies of species of Basidiobolus and other medically important fungi. J Clin Microbiol 1986; 34: 679-82. 9. Thianprasit M. Cutaneous Pythiosis in Human-Being. Abstract in Siriraj 100th Academic Meeting, Mahidol University. Bangkok, Thailand, 18-22 April 1988: 94. i0. Kaufman L, Mendoza L, Standard PG. Immunodiffusion test for serodiagnosing subcutaneous zygomycosis. J Clin Microbiol 1990; 28: 1887-1890.
