Mycopathologia 115: 1-8, 1991. 9 1991 KluwerAcademic Publishers. Printed in the Netherlands.
Rhinofacial zygomycosis caused by Conidiobolum coronatus A case r e p o r t
Alcyr Ribeiro Costa, 1 Edward Porto, 2 Jos6 Roberto Pereira Pegas, 3 Vitor Manuel Silva dos Reis, 3 Mario Cezar Pires, 3 Carlos da Silva Lacaz, 2 Maria Conceig~io Rodrigues, 4 Helena Mtiller 3 & Luis Carlos Cuc65 1Hospital do Servidor P(tblico Estadual 'Francisco Morato de Oliveira', Servifo de Dermatologia; 2Laborat6rio de Micologia M6dica, Instituto de Medicina Tropical de S6o Paulo e Laboratdrio de Investigaf~o Mddica, No. 53 - Hospital das Clfnicas, Faculdade de Medicina da Univesidade de S6o Paulo, Brasil; 3Cornplexo Hospitalar de Guarulhos, S6o Paulo; 4Divis~o de Tisiologia e Pneumologia Sanitdria, Laborat6rio de Micologia Medica, Instituto Clemente Ferreira, S6o Paulo; 5Hospital das Chnicas, Faculdade de Medicina da Universidade de S6o Paulo, Servifo de Dermatologia Received 24 July 1990; accepted in revised form 27 February 1991
Key words: Conidiobolus coronatus, Brazil, microconidiophores, multiplicative conidia, papilla, zygomycosis
Abstract
A case of rhinofacial zygomycosis with of years duration, caused by Conidiobolus coronatus is described. The patient, a 72-years-old woman, presented with a bilateral distortion of the subcutaneous tissue and disfigurement of the face. Treatment with ketoconazole and potasium iodide did not prevent several relapses. At present she is still under treatment with fluconazole with clinical healing. Histopathological and mycological examination confirmed the dermatological diagnosis. An increasing number of cases of zygomycosis caused by fungi of the order Entomophthorales have also been reported in the Northern and Northeastern States of Brazil.
Introduction
Basidiobolus ranarum Eidem 1886 (Synonymy B. haptosporus), Conidiobolus coronatus (Costantin) Batko 1964, and C. incongruus Drechsler 1960 are the only species of the Entomophthorales that are known to cause human disease [1]. C. lamprauges infection in a horse has been reported once [2]. The valid species of the genus Basidiobolus and Conidiobolus, class Zygomycetes order Entomophthorales belong to the family Entomophthora-
ceae [1]. There are 27 recognized species in the key to the genus Conidiobolus [1]. C. coronatus occurs as saprophyte and is specially abundant in plant detritus [3, 4]. However, it causes disease in humans only in the warmer regions of the world. In addition to infecting humans, C. coronatus also infects other mammals and insects over a broader geographical range than that in which it infects humans. C. incongruus has been isolated only in the United States: twice from plant detritus and once from humans [1, 5]. King [1] placed C. coronatus among the
most abundant and cosmopolitan of the Conidiobolus species. All steps of the morphological cycle of Entomophthora coronata (= Conidiobolus coronatus) on Sabouraud-dextrose agar were described [6] and the systematics of C. coronatus have been recently considered in detail [1, 4, 5]. In Brazil, from March to December of 1977, 124 samples taken from guts and dungs of 13 different sepcies of amphibians and reptdes yielded 64 isolates belonging to the specie Basidiobolus haptosporus (=B. ranarum). For the first time in Brazil this entomophthoraceous is reported as occurring in reptiles and amphibians [7]. During a five-months period Porto et al. [8] collected 160 soil samples, with and without plant detritus from different cities in Brazil. Two of these 160 samples (1.25%) yielded C. coronatus in culture. A case of zygomycosis in mule caused by Entomophthora coronata was reported [9]. There are several reports in the specialized literature of cases of rhinofacial zygomycosis caused by C. coronatus from the tropical states of Piaui, Maranh~o and Bahia in Brazil. All these cases present clinical and histopathological documentation but only a few cases showed a mycological diagnosis [10, 11]. An excellent review of entomophthoromycosis caused by B. haptosporus and C. coronatus in Brazil was recently published [12]. The form of entomophthoromycosis centerfacial, visceral or systemic is generally caused by C. coronatus and subcutaneous form by B. haptosporus. Until recently some 200 or more human infections by Basidiobolus and Conidiobolus species, order Entomophthorales, occurred in otherwise healthy people. Now these organisms are appearing in the more general opportunistic setting [13]. The authors report a case of rhinofacial zygomycosis caused by C. coronatus with lesions and bilateral distortion of the subcutaneous tissue and disfigurement of the overlaying tissues. The patient, from the Northern State of Maranhfto,
ATLANTIC CEAN
N
S Fig. 1. Location of S~o Luiz (SL), the patients residence.
worked in farm lands where she was probably hurt and infected by the entomophthoraceous present in plant detritus. Histopathological and mycological findings confirmed the diagnosis.
Case report M.H.C., 72-years-old, white female, worked in farm lands in Silo Lufz do Maranhgo (Fig. 1), Northern State of Brazil. Five years ago she presented with a nasal obstruction. After six months she observed a lump inside the nose. The lesion was excised in a local clinic, with no pathological or mycological examinations9 After 10 months she again presented herself with nose, lips and frontal face edema. In Rio de Janeiro a biopsy of the lesion was performed. With a referred diagnosis of mycosis, treatment was started with ketoconazole, 400 mg per day, for one year, with partial healing of the lesions. Nearly two years ago the lesions reappeared and ketoconazole was given again (Figs9 2a and 2b). After 6 months the patient was referred to the 'Complexo Hospitalar de Guarulhos - Dermatologia'. Ketoconazole was again administered 400 mg per day, for 4 months. The clinical material obtained from the
cological (imprint), immunoperoxidase (IgG, IgA, IgM) in tissue, serology for leishmaniasis and paracoccidioidomycosis presented negative results. The tests for cellular immunity PPD, streptokinase, candidin, paracoccidioidin, Mitsuda and Escherichia coli were also negative.
Histopathological findings
Fig. 2. (a) Rhinofacial zygomycosis caused by Conidiobolus coronatus with lesions and bilateral distortion of the subcutaneous tissue with disfigurement of the overlaying tissues; (b) Patient under treatment with fluconazole.
Histological sections stained by haematoxylin and eosin, and Gomori-methenamine silver associate to Mayer's carmalum showed a mucous membrane coated by stratified squamous epithelium. A pyogranulomatous inflammatory reaction was seen in tissue with an infiltrate of monocytes, neutrophils, eosinophils, epithelioid cells and giant cells of Langhans type. The center of this area showed hyphae transverse and longitudinally sectioned without cytoplasm, surrounded by typical eosinophilic radiate material of SplendoreHoeppli (Fig. 3a). This material was also stained in dark-red by the Mayer's carmalum method (Fig. 3b). The hyphae were sparsely to regularly septate. The histopathological pattern is indicative of 'rhinofacial zygomycosis' caused by a fungus of the order Entomophthorales. This diagnosis was confirmed by the isolation and identification of Conidiobolus coronatus from biopsy of the lesions.
Materials and methods biopsy of the lesions was sent to the laboratory for mycological and histopathological examinations. At present the patient is under treatment with fluconazole, 200 mg per day, with clinical healing and no relapses.
Laboratory examinations
Biopsy material of the lesion was cultured on Sabouraud-dextrose agar. After nearly a week the cultures showed colonies of the light buff color growing together with dark brown colonies of dematiaceous fungus. The isolation of Conidiobolus coronatus from the contaminated culture was performed by the use of the following steps:
Hemogram, billirubin, proteinogram, glycemia, urine type I, urea and creatinin had normal values. VDRL, baciloscopy of the ear lobulus, my-
a) Obtaining pure colonies. (1) 18 ml of potato dextrose agar were poured in the inferior part of a Petri dish and left to solidify. 2-3 ml of the same
Fig. 3. Histological sections: (a) Haematoxylin and eosin stain showing hypha of Conidiobolus coronatus in oblique section, without cytoplasm,surrounded by eosinophilicmaterial of Slendore-Hoeppli. x 190; (b) Transversal sectioned hypha; longitudinal section of a hypha with evident septum. Gomori-methenaminesilver associated to Mayer's carmalum method, x 190.
medium were placed in the upper part yielding a circle of nearly 3.5 cm of diameter after solidification. (2) Little portions of the culture of C. coronatus contaminated with C l a d o s p o r i u m sp. were seeded on the plate. The Petri dish was inverted at room temperature and natural light. After nearly 48 hours, several pure colonies were formed by the expelling of the conidia to the medium in the cover of the dish. The medium in the inferior part of the dish also showed several satellite colonies of light buff color. Later the culture was covered by a powdery pellicle, due to the germination of many expelled conidia. Colonies of C l a d o s p o r i u m sp. were again observed in the inferior part of the dish. (3) Little pieces of the pure culture of the lid of the dish were
transferred to slant tubes containing potato dextrose agar.
b) Slide culture. (1) One of two slides of potato dextrose agar was seeded with spores of C. coronatus. (2) The upper slide was placed in an inverted position so the surface with the culture medium would receive the expelled spores from the growth on the inferior slide. The wet chamber was maintained at room temperature and natural light. (3) The slide culture was stained by the cotton blue method of Porto et al. [14]. The various phases of C. coronatus were documented in optical and scanning microscopy (Figs. 4 and 5).
Fig. 4. Slide culture of C. coronatus stained by cotton-blue: (a) Globose primary conidia in development and maturation in the apex of the unbranched conidiophores; two of them presenting papillae (arrows). x210; (b) Primary conidium with papilla and granular cytoplasm born on unbranched conidiophore and discharged conidium with rounded shape of papilla. • (c and d) Discharged primary conidium in the center with short conidiophore and the secondary one formed it on its apex. Conidium with two multiplicative conidia or microspores at upper left and the same conidium with more microspores at smaller magnification (inset) at upper right. At lower part discharged conidium with papilla and three short appendages (arrows). (c) x 340; (d) x 210.
conidia or microspores were formed on short microconidiophores. Only a few primary conidia and a few short microconidiophores showed multiplicative conidia. A few typical villose resting conidia with many long microconidiophores (villose appendages) were found in the culture of biopsy material of this case (Figs. 4c and 4d).
Discussion
Fig. 5. Conidiophore, primary and secondary conidium. A few conidia with papillae can be observed. Scanning electron microscopy x 2100.
Mycological findings a) Macroscopic aspect. The fungus grew on potato dextrose agar in 24-48 hours at room temperature and daylight exposure. Several satellite colonies of light buff color were formed. The culture was later covered by a powdery pellicle due to the germination of many expelled conidia. b) Micromorphology. Slide culture on potato dextrose agar stained with cotton-blue [14], and showed globose primary conidia with an-unbranched conidiophores (sporangiophores) arising from vegetative mycelium (Figs. 4a and 4b). The discharged primary conidium or sporangiolum formed short conidiophore and the secondary one formed it on its apex. From the discharged primary conidium the multiplicative
Over 150 cases of rhinofacial zygomycosis have been recorded from humans. The etiologic agent, in cases in which cultural identification has been obtained was Conidiobolus coronatus. Most human cases have occurred in Central and West Africa. A few cases have also been recorded in Colombia, Brazil and the Caribbean [13]. C. coronatus generally causes a chronic infection of the subcutaneous tissue and submucosa of the nose and face. This zygomycete is found worldwide but it is most abundant in tropical areas. The first human case was described in Jamaica in 1965 [15]. The clinical description of the present case is in agreement with those reported in the specialized literature in Brazil. The patient presented facial tumefaction, nasal obstruction, skin, nose and upper lip infiltration, with typical face deformation. The histological picture of the infection caused by C. coronatus or B. haptosporus is identical. Morphological differentiation was performed by the authors in slide culture. With basis in the 'Key to species of Conidiobolus' [4] the zygomycete was identified according to King [1] from the following micromorphological characteristics: (a) microconidia produced, (b) villose resting spores produced, (c) discharged primary conidium with rounded shape of papillum, and (d) zygospores not produced. Conidiobolus incongruus presents different characteristics from C. coronatus: (a) villose resting spores not produced, (b) discharged primary conidium with pointed shape papillum, (c) mature zygospore present, and (d) hyphal seg-
ments becoming distended in older portions of colony. C. incongruus is rarely isolated from nature and has been identified as the etiologic agent of rhinofacial zygomycosis in only a few cases. Villose conidia are produced only by C. coronatus with the production of a thick wall and growth of villose appendages. The appendages are quite variable in number and length [1]. Most strains of C. corouatus readily produce villose conidia but their production is difficult to induce in a small percentage of strains. This appears to be particularly true for human isolates [16]. In the present case the patient was treated with increasing doses of potassium iodide up to the maximum of 2 g per day during a month without good results. This drug was suspended and replaced by ketoconazole 400rag per day during four months with slight healing and no progression. At present the patient is still under treatment with fluconazole, 200 mg per day, with clinical healing and no relapses. No single drug has proven to be effective in all cases of zygomycosis caused by C. coronatus. These clinical data, plus few reports of in vitro sensitivity to antifungal agents suggest that there is considerable strain variability in sensitivity (17). Amphothericin B has been recommended in patients that do not respond to potassium iodide therapy [13]. In the past, many cases of rhinofacial zygomycosis proven to be caused by C. coronatus showed that this fungus was resistant to the drugs then used in the treatment [18]. In the present case the authors did not use amphothericin B, but obtained good healing of the patient with ketoconazole and better results with fluconazole. The authors emphasize the value of the early diagnosis and treatment of rhinofacial zygomycosis due to the deep deformation of the face. C. coronalus has the potential capacity to reach by adjacency the neck and other structures, increasing the severity of the infection or leading to decrease.
Acknowledgements The authors are grateful to Dr. Flavio P. Faria and Prof. Antonio Sesso from the Departamento de Patologia da Faculdade de Medicina da Universidade de Silo Paulo, for the scanning electron photomicrography.
References 1. King DS. Entomophthorales. In: Howard DH, Howard LF, eds. Fungi Pathogenic for Humans and Animals, Part A: Biology. New York: Marcel Dekker 1983: 61-72. 2. Humber RA, Brown CC. The first report of Conidiobolus lamprauges from a vertebrate rhinoconidiobolomycosis. Mycol Soc Am Abstracts 1987; 6; 11. 3. Drechsler, C. Widespread distribution of Delacroixia coronata and other saprophytic Entomophthoraceae in plant detritus. Science 1952; 115: 575-6. 4. King DS. Systematics of Conidiobolus (Entomophthorales) using numerical taxonomy, I: biology and cluster analysis. Can J Bot 1976; 54: 45-65. 5. King DS. Systematics of Conidiobolus (Entomophthrales) using numerical taxonomy, III: descriptions of recognized species. Can J Bot 1977; 55; 718-29. 6. Praserthon S. Conidial formation in Entornophthora coronata (Costantin) Kevorkian. J Insect Pathol 1963; 5: 318-35. 7. Porto E, Milanez AI. Basidiobolus isolados de r4pteis e anffcios no Brasil. Rev Inst Med trop S~o Paulo 1979; 21: 237-45. 8. Porto E, Melo NT, Heins-Vaccari EM, Lacaz C da S, Assis CM. Isolamento de Conidiobolus coronatus (Costantin) Batko 1964, de amostras de terra com e sem detritus vegetais. An bras Dermatol 1987; 62: 303-7. 9. Johnston MJ, Soerensen B, Saliba AM, Lacaz C da S, Bela Neto J, Cruz JM. Isolamento de Entomophthora coronata. Arq Inst Biol. 1967; 34: 51-8. 10. Towersey, L, Wanke B, Estrella RR, Londero AT, Mendonga AMN, Neves RG. Conidiobolus coronatus infection treated with ketoconazole. Arch Dermatol 1988; 124: 1392-6. 11. Fonseca APM, Fonseca WSM, Leal MJS, Arafijo RC. Rinoentomoftoromicose: relato quatro casos. An bras Dermatol 1989; 64: 261-5. 12. Bittenconrt AL. Entomoftoromicose. Revis~o. Med Cut ILA 1988; 16: 93-100. 13. Rippon JW. Zygomycosis. In: Rippon JW. Medical Mycology: The Pathogenic Fungi and the Pathogenic Actinomycetes, 3rd ed. Philadelphia: WB Saunders Company 1988: 687-699. 14. Porto E, Takahashi N, Heins EM, Lacas C da S. Nuevo metodo para microcultivo de hongos. Rev Arg Micologia 1981; 4: 24-9.
15. Bras G, Gordon CC, Emmons CW, Brendegast KM, Sugar M. A case of phycomycosis observed in Jamaica: infection with Entornophthora coronata. Am J Trop Med Hyg 1965; 14: 141-5. 16. King DS. Systematics of fungi causing entomophthoromycosis. Mycologia 1979; 71: 731-45. 17. Taylor GD, Sehkon AS, Tyrrell DLJ, Goldsand G. Rhinofacial zygomycosis caused by Conidiobolus coronatus: a case report including in vitro sensitivity to antimycotic agents. Am J Trop Med Hyg 1987; 36: 398-401.
18. Williams AO. Pathology of phycomycosis due to Entomophthora and Basidiobolus species. Arch Pathol Lab Med 1969; 87: 13-20. Address for correspondence: Prof. Dr. Carlos da Silva Lacaz Rua Jos6 Maria Lisboa, 558-5 ~ Andar (Jardim Paulista), CEP-01423 S~o Paulo, Brasil
Rhinofacial zygomycosis caused by Conidiobolum coronatus A case r e p o r t
Alcyr Ribeiro Costa, 1 Edward Porto, 2 Jos6 Roberto Pereira Pegas, 3 Vitor Manuel Silva dos Reis, 3 Mario Cezar Pires, 3 Carlos da Silva Lacaz, 2 Maria Conceig~io Rodrigues, 4 Helena Mtiller 3 & Luis Carlos Cuc65 1Hospital do Servidor P(tblico Estadual 'Francisco Morato de Oliveira', Servifo de Dermatologia; 2Laborat6rio de Micologia M6dica, Instituto de Medicina Tropical de S6o Paulo e Laboratdrio de Investigaf~o Mddica, No. 53 - Hospital das Clfnicas, Faculdade de Medicina da Univesidade de S6o Paulo, Brasil; 3Cornplexo Hospitalar de Guarulhos, S6o Paulo; 4Divis~o de Tisiologia e Pneumologia Sanitdria, Laborat6rio de Micologia Medica, Instituto Clemente Ferreira, S6o Paulo; 5Hospital das Chnicas, Faculdade de Medicina da Universidade de S6o Paulo, Servifo de Dermatologia Received 24 July 1990; accepted in revised form 27 February 1991
Key words: Conidiobolus coronatus, Brazil, microconidiophores, multiplicative conidia, papilla, zygomycosis
Abstract
A case of rhinofacial zygomycosis with of years duration, caused by Conidiobolus coronatus is described. The patient, a 72-years-old woman, presented with a bilateral distortion of the subcutaneous tissue and disfigurement of the face. Treatment with ketoconazole and potasium iodide did not prevent several relapses. At present she is still under treatment with fluconazole with clinical healing. Histopathological and mycological examination confirmed the dermatological diagnosis. An increasing number of cases of zygomycosis caused by fungi of the order Entomophthorales have also been reported in the Northern and Northeastern States of Brazil.
Introduction
Basidiobolus ranarum Eidem 1886 (Synonymy B. haptosporus), Conidiobolus coronatus (Costantin) Batko 1964, and C. incongruus Drechsler 1960 are the only species of the Entomophthorales that are known to cause human disease [1]. C. lamprauges infection in a horse has been reported once [2]. The valid species of the genus Basidiobolus and Conidiobolus, class Zygomycetes order Entomophthorales belong to the family Entomophthora-
ceae [1]. There are 27 recognized species in the key to the genus Conidiobolus [1]. C. coronatus occurs as saprophyte and is specially abundant in plant detritus [3, 4]. However, it causes disease in humans only in the warmer regions of the world. In addition to infecting humans, C. coronatus also infects other mammals and insects over a broader geographical range than that in which it infects humans. C. incongruus has been isolated only in the United States: twice from plant detritus and once from humans [1, 5]. King [1] placed C. coronatus among the
most abundant and cosmopolitan of the Conidiobolus species. All steps of the morphological cycle of Entomophthora coronata (= Conidiobolus coronatus) on Sabouraud-dextrose agar were described [6] and the systematics of C. coronatus have been recently considered in detail [1, 4, 5]. In Brazil, from March to December of 1977, 124 samples taken from guts and dungs of 13 different sepcies of amphibians and reptdes yielded 64 isolates belonging to the specie Basidiobolus haptosporus (=B. ranarum). For the first time in Brazil this entomophthoraceous is reported as occurring in reptiles and amphibians [7]. During a five-months period Porto et al. [8] collected 160 soil samples, with and without plant detritus from different cities in Brazil. Two of these 160 samples (1.25%) yielded C. coronatus in culture. A case of zygomycosis in mule caused by Entomophthora coronata was reported [9]. There are several reports in the specialized literature of cases of rhinofacial zygomycosis caused by C. coronatus from the tropical states of Piaui, Maranh~o and Bahia in Brazil. All these cases present clinical and histopathological documentation but only a few cases showed a mycological diagnosis [10, 11]. An excellent review of entomophthoromycosis caused by B. haptosporus and C. coronatus in Brazil was recently published [12]. The form of entomophthoromycosis centerfacial, visceral or systemic is generally caused by C. coronatus and subcutaneous form by B. haptosporus. Until recently some 200 or more human infections by Basidiobolus and Conidiobolus species, order Entomophthorales, occurred in otherwise healthy people. Now these organisms are appearing in the more general opportunistic setting [13]. The authors report a case of rhinofacial zygomycosis caused by C. coronatus with lesions and bilateral distortion of the subcutaneous tissue and disfigurement of the overlaying tissues. The patient, from the Northern State of Maranhfto,
ATLANTIC CEAN
N
S Fig. 1. Location of S~o Luiz (SL), the patients residence.
worked in farm lands where she was probably hurt and infected by the entomophthoraceous present in plant detritus. Histopathological and mycological findings confirmed the diagnosis.
Case report M.H.C., 72-years-old, white female, worked in farm lands in Silo Lufz do Maranhgo (Fig. 1), Northern State of Brazil. Five years ago she presented with a nasal obstruction. After six months she observed a lump inside the nose. The lesion was excised in a local clinic, with no pathological or mycological examinations9 After 10 months she again presented herself with nose, lips and frontal face edema. In Rio de Janeiro a biopsy of the lesion was performed. With a referred diagnosis of mycosis, treatment was started with ketoconazole, 400 mg per day, for one year, with partial healing of the lesions. Nearly two years ago the lesions reappeared and ketoconazole was given again (Figs9 2a and 2b). After 6 months the patient was referred to the 'Complexo Hospitalar de Guarulhos - Dermatologia'. Ketoconazole was again administered 400 mg per day, for 4 months. The clinical material obtained from the
cological (imprint), immunoperoxidase (IgG, IgA, IgM) in tissue, serology for leishmaniasis and paracoccidioidomycosis presented negative results. The tests for cellular immunity PPD, streptokinase, candidin, paracoccidioidin, Mitsuda and Escherichia coli were also negative.
Histopathological findings
Fig. 2. (a) Rhinofacial zygomycosis caused by Conidiobolus coronatus with lesions and bilateral distortion of the subcutaneous tissue with disfigurement of the overlaying tissues; (b) Patient under treatment with fluconazole.
Histological sections stained by haematoxylin and eosin, and Gomori-methenamine silver associate to Mayer's carmalum showed a mucous membrane coated by stratified squamous epithelium. A pyogranulomatous inflammatory reaction was seen in tissue with an infiltrate of monocytes, neutrophils, eosinophils, epithelioid cells and giant cells of Langhans type. The center of this area showed hyphae transverse and longitudinally sectioned without cytoplasm, surrounded by typical eosinophilic radiate material of SplendoreHoeppli (Fig. 3a). This material was also stained in dark-red by the Mayer's carmalum method (Fig. 3b). The hyphae were sparsely to regularly septate. The histopathological pattern is indicative of 'rhinofacial zygomycosis' caused by a fungus of the order Entomophthorales. This diagnosis was confirmed by the isolation and identification of Conidiobolus coronatus from biopsy of the lesions.
Materials and methods biopsy of the lesions was sent to the laboratory for mycological and histopathological examinations. At present the patient is under treatment with fluconazole, 200 mg per day, with clinical healing and no relapses.
Laboratory examinations
Biopsy material of the lesion was cultured on Sabouraud-dextrose agar. After nearly a week the cultures showed colonies of the light buff color growing together with dark brown colonies of dematiaceous fungus. The isolation of Conidiobolus coronatus from the contaminated culture was performed by the use of the following steps:
Hemogram, billirubin, proteinogram, glycemia, urine type I, urea and creatinin had normal values. VDRL, baciloscopy of the ear lobulus, my-
a) Obtaining pure colonies. (1) 18 ml of potato dextrose agar were poured in the inferior part of a Petri dish and left to solidify. 2-3 ml of the same
Fig. 3. Histological sections: (a) Haematoxylin and eosin stain showing hypha of Conidiobolus coronatus in oblique section, without cytoplasm,surrounded by eosinophilicmaterial of Slendore-Hoeppli. x 190; (b) Transversal sectioned hypha; longitudinal section of a hypha with evident septum. Gomori-methenaminesilver associated to Mayer's carmalum method, x 190.
medium were placed in the upper part yielding a circle of nearly 3.5 cm of diameter after solidification. (2) Little portions of the culture of C. coronatus contaminated with C l a d o s p o r i u m sp. were seeded on the plate. The Petri dish was inverted at room temperature and natural light. After nearly 48 hours, several pure colonies were formed by the expelling of the conidia to the medium in the cover of the dish. The medium in the inferior part of the dish also showed several satellite colonies of light buff color. Later the culture was covered by a powdery pellicle, due to the germination of many expelled conidia. Colonies of C l a d o s p o r i u m sp. were again observed in the inferior part of the dish. (3) Little pieces of the pure culture of the lid of the dish were
transferred to slant tubes containing potato dextrose agar.
b) Slide culture. (1) One of two slides of potato dextrose agar was seeded with spores of C. coronatus. (2) The upper slide was placed in an inverted position so the surface with the culture medium would receive the expelled spores from the growth on the inferior slide. The wet chamber was maintained at room temperature and natural light. (3) The slide culture was stained by the cotton blue method of Porto et al. [14]. The various phases of C. coronatus were documented in optical and scanning microscopy (Figs. 4 and 5).
Fig. 4. Slide culture of C. coronatus stained by cotton-blue: (a) Globose primary conidia in development and maturation in the apex of the unbranched conidiophores; two of them presenting papillae (arrows). x210; (b) Primary conidium with papilla and granular cytoplasm born on unbranched conidiophore and discharged conidium with rounded shape of papilla. • (c and d) Discharged primary conidium in the center with short conidiophore and the secondary one formed it on its apex. Conidium with two multiplicative conidia or microspores at upper left and the same conidium with more microspores at smaller magnification (inset) at upper right. At lower part discharged conidium with papilla and three short appendages (arrows). (c) x 340; (d) x 210.
conidia or microspores were formed on short microconidiophores. Only a few primary conidia and a few short microconidiophores showed multiplicative conidia. A few typical villose resting conidia with many long microconidiophores (villose appendages) were found in the culture of biopsy material of this case (Figs. 4c and 4d).
Discussion
Fig. 5. Conidiophore, primary and secondary conidium. A few conidia with papillae can be observed. Scanning electron microscopy x 2100.
Mycological findings a) Macroscopic aspect. The fungus grew on potato dextrose agar in 24-48 hours at room temperature and daylight exposure. Several satellite colonies of light buff color were formed. The culture was later covered by a powdery pellicle due to the germination of many expelled conidia. b) Micromorphology. Slide culture on potato dextrose agar stained with cotton-blue [14], and showed globose primary conidia with an-unbranched conidiophores (sporangiophores) arising from vegetative mycelium (Figs. 4a and 4b). The discharged primary conidium or sporangiolum formed short conidiophore and the secondary one formed it on its apex. From the discharged primary conidium the multiplicative
Over 150 cases of rhinofacial zygomycosis have been recorded from humans. The etiologic agent, in cases in which cultural identification has been obtained was Conidiobolus coronatus. Most human cases have occurred in Central and West Africa. A few cases have also been recorded in Colombia, Brazil and the Caribbean [13]. C. coronatus generally causes a chronic infection of the subcutaneous tissue and submucosa of the nose and face. This zygomycete is found worldwide but it is most abundant in tropical areas. The first human case was described in Jamaica in 1965 [15]. The clinical description of the present case is in agreement with those reported in the specialized literature in Brazil. The patient presented facial tumefaction, nasal obstruction, skin, nose and upper lip infiltration, with typical face deformation. The histological picture of the infection caused by C. coronatus or B. haptosporus is identical. Morphological differentiation was performed by the authors in slide culture. With basis in the 'Key to species of Conidiobolus' [4] the zygomycete was identified according to King [1] from the following micromorphological characteristics: (a) microconidia produced, (b) villose resting spores produced, (c) discharged primary conidium with rounded shape of papillum, and (d) zygospores not produced. Conidiobolus incongruus presents different characteristics from C. coronatus: (a) villose resting spores not produced, (b) discharged primary conidium with pointed shape papillum, (c) mature zygospore present, and (d) hyphal seg-
ments becoming distended in older portions of colony. C. incongruus is rarely isolated from nature and has been identified as the etiologic agent of rhinofacial zygomycosis in only a few cases. Villose conidia are produced only by C. coronatus with the production of a thick wall and growth of villose appendages. The appendages are quite variable in number and length [1]. Most strains of C. corouatus readily produce villose conidia but their production is difficult to induce in a small percentage of strains. This appears to be particularly true for human isolates [16]. In the present case the patient was treated with increasing doses of potassium iodide up to the maximum of 2 g per day during a month without good results. This drug was suspended and replaced by ketoconazole 400rag per day during four months with slight healing and no progression. At present the patient is still under treatment with fluconazole, 200 mg per day, with clinical healing and no relapses. No single drug has proven to be effective in all cases of zygomycosis caused by C. coronatus. These clinical data, plus few reports of in vitro sensitivity to antifungal agents suggest that there is considerable strain variability in sensitivity (17). Amphothericin B has been recommended in patients that do not respond to potassium iodide therapy [13]. In the past, many cases of rhinofacial zygomycosis proven to be caused by C. coronatus showed that this fungus was resistant to the drugs then used in the treatment [18]. In the present case the authors did not use amphothericin B, but obtained good healing of the patient with ketoconazole and better results with fluconazole. The authors emphasize the value of the early diagnosis and treatment of rhinofacial zygomycosis due to the deep deformation of the face. C. coronalus has the potential capacity to reach by adjacency the neck and other structures, increasing the severity of the infection or leading to decrease.
Acknowledgements The authors are grateful to Dr. Flavio P. Faria and Prof. Antonio Sesso from the Departamento de Patologia da Faculdade de Medicina da Universidade de Silo Paulo, for the scanning electron photomicrography.
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