EUROPEAN
Eur. J. Epidemiol. 0392-2990
Vol. 8, No. 3
JOURNAL
May 1992, p. 397-406
OF
EPIDEMIOLOGY
PROTOTHECOSIS: A REPORT OF TWO CASES IN JAPAN AND A REVIEW OF THE LITERATURE T. MATSUDA* and T. MATSUMOTO *.1 *Department o f Dermatology - FacuRy o f Medicine - Kyushu University - Fukuoka - Japan. **Department o f Dermatology - Toshiba General Hospital - 6-3-22 Higashi-oi - Shinagawa-ku Tokyo - Japan.
Key words: Prototheca - Protothecosis - Human cases Protothecosis is an emerging opportunistic infection caused by species belonging to the genus Prototheca. Two Japanese cases of protothecosis are documented with a critical review of the
literature. A current perspective concerning the microbiology and disease entity of protothecosis is described in detail.
INTRODUCTION
Protothecosis is a relatively rare infection caused by the species of the achlorophyllic genus Prototheca Krfiger, microorganisms morphologically similar to those of the genus Chlorella. The cases of protothecosis in human have been classified into the three clinical forms: 1) cutaneous and/or subcutaneous infection, 2)synovitis of olecranon bursa or other fibrous tissue, and 3) systemic infection. Recently, the disease has become very important both to physicians and microbiologists because of the increasing number of the reported cases, characteristic clinical features, and a distinct morphology of the etiologic agents in vitro as well as in vivo. The purposes of this presentation are to describe two Japanese cases of protothecosis, and to review and discuss of the literature of human protothecosis. Report of the cases Case 1.
An 80-year-old Japanese male patient noticed a spontaneous, asymptomatic red papule on his right 1 Corresponding author.
antecuboidal fossa in March 1987. He did not have any apparent antecedent traumatic history at the site of the lesion. He visited his neighbor physician, who diagnosed the lesion as chronic eczema. In spite of topical corticosteroid application for a couple of months, the lesion gradually spread, and he developed another extending plaque on his left arm. He consulted to the Dermatology Clinic, Kyushu University Hospital in August 1987. On physical examination, irregularly shaped, well demarcated, erythematous, infiltrated plaques are distributed symmetrically on the flexor surface of his arms. The surface of the lesions were slightly elevated and accompanied with vesicles, pustules, crusts, and fine scale (Fig. 1). No regional lymph nodes were palpable. He is a retired rice farmer in Kyushu island, the south-western area of Japan, and occasionally enters the rice paddy. Laboratory investigation revealed that he had an untreated diabetes and hyperglycemia. No immunological abnormality was detected. A skin biopsy specimen taken from a plaque on his right upper arm, stained with hematoxylin and eosin was histologically examined. In the dermis, there revealed a dense, granulomatous infiltration consisted mainly of lymphocytes, histiocytes, and scattered foreign body giant cells. Small, hyaline,
397
Matsuda T. and Matsumoto T.
Eur. J. Epidemiol.
Figure 1. - Clinical features of Case 1. Symmetrically distributed erythematous plaques on upper extremities.
round and yeast-like cells and large spherical cells were abundantly recognized within the multinuclear giant cells or in the interstitial tissue of the middle to reticular dermis. Further detailed observation of the microorganisms was performed with periodic acidSchiff staining. Small and single cells contained a large and single nucleus. Large cells were divided by nuclear and cytoplasmic cleveages, and appeared like wheels. Very large cells, containing 4-20 endospores, showed morula-like appearance (Fig. 2). Several pieces from a biopsy specimen were inoculated on Sabouraud dextrose agar plates and incubated at 25° C. From each of these, identical colonies were obtained in pure culture. The isolated organism yielded rapidly growing, smooth, moist, and creamy colonies. Microscopically, single cells were hyaline, globose to ovoid, and variable in size, measuring 3 to 12 lain in diameter. Some of the larger components were divided by cleavages, and endospores were contained within the others (Fig. 3). The mother cells were ruptured to release the spores, then each spore increased in size and eventually became mother cell. The isolate grew well at 37° C, and failed to hydrolyze urea. Assimilation test by the API 20 system showed positive results with galactose,
glucose, glycerol, and trehalose. On the basis of morphologic and physiologic characteristics, the isolate was identified as Prototheca wickerhamii Tubaki et Soneda. Immunohistopathologic study by anti-P. wickerhamii globulin, which was performed at the :Centers for Diseases Control's Division of Mycotic Diseases, also confirmed this identification. The patient was treated with oral administration of ketoconazole, a daily dose of 200 rag. Clinical effect appeared rapidly as the plaques discolored and the induration decreased. After 19 weeks of chemotherapy, each lesions healed leaving superficial scars. A biopsy specimen taken after 17 week ketaconazole therapy revealed no distinct microorganisms. The patient showed no recurrence during 3 year follow-up period, as of May 1991. Case 2.
A 13-year-old Japanese boy complained of general fatigue and palpitation in December 1987. He admitted to Hamanomachi Hospital in Fukuoka and was diagnosed as a patient with iron deficiency anemia due to gastrointestinal bleedings from unknown lesions. In October 1988, he developed a high fever
398
Vol. 8, 1992
Protothecosis from Japan
Figure 2. - Biopsy specimen showing spherical and wheel-like morphologies of etiologic agent accompanied by granulomatous inflammation (periodic acid-Schiff stain).
Figure 3. - Isolated microorganism showing various stages of its life cycle (wet preparation in lactophenol cotton blue).
399
Matsuda T. and Matsumoto T.
Eur. J. Epidemiol.
and severe general fatigue. Hepatosplenomegaly, liver abscesses, and enlarged abdominal lymph nodes were detected by means of body CT and upper abdominal echogram. On exploratory laparotomy, multiple, whitecolored, and small nodules were observed both in the submucosa of small intestine and on the liver surface. Mesenteric and ileocecal lymph nodes were swollen. Incisional biopsies were performed on each of these lesions. The white nodules in submucosa were revealed to be consisted mainly of Prototheca cells, which were surrounded by the dense inflammatory cellular infiltration (Fig. 4). In lymph nodes, abundant Prototheca cells were also recognized (Fig. 5). The isolation of the etiologic agent from stool materials were unsuccessful. However, the causative microorganism was later isolated from meningeal aspirate and identified as P. wickerhamii Tubaki et Soneda. Immunofluorescent study performed at the Centers for Disease Control's Division of Mycotic Disease also confirmed the identification. Intravenous amphotericin B injection for 11 weeks (total dose 1230 mg) was performed. On week 5
the patient developed headaches and double vision. Protothecal meningitis was suspected and intrathecal amphotericin B injection was also executed for 16 weeks (total dose 9.25 mg). The second laparotomy performed on week 23 revealed no significant lesion.
Figure 4. - Specimen form a submucosal nodule on intestine, consisted mainly of Prototheca cells (periodic acid-Schiff stain),
Figure 5. - Specimen from abdominal lymph node showing numerous Prototheca cells (periodic acid-Schiff stain).
Comment
Etiology The Prototheca species are achlorophyllic microorganisms morphologically similar to the green alga in the genus Chlorella. Prototheca zopfii, the type strain of the genus Prototheca, was originally isolated from the slime flux of trees by Krtiger in 1894 (22). Subsequent studies aroused a long lasting controversy concerning of the taxonomic position, alga or fungus, of this genus. Currently, the genus Prototheca is classified in alga on the basis of morphologic characteristics, life cycle, and reproduction system, common antigenic nature, and especially of the ultrastructural findings (34, 42). Morphologically, the Prototheca species grow rapidly on routine laboratory media without
400
Vol. 8, 1992
Protothecosis from Japan
cycloheximide. Colonies are soft, wet, yeast-like, and white to light tan in color. Microscopic examination reveals globose to ovoid hyaline cells measuring 1.3-16 lain in diameter. Each cell enlarges in size and makes nuclear division several times, followed by cleavage of the cytoplasm. Mature sporangium, containing 2 to 20 or more auto(endo) spores, ruptures and releases daughter cells. This reproductive cycle is repeated. Mycelium and conidia are absent. Species of the genus Prototheca are commonly isolated from various natural sources, such as slime flux of trees, soil, detritus, animal waste, and sewege (41). Four Prototheca species are currently recognized: P. salmonis Gentles et Bond 1977, P. stagnora W.B. Cooke 1968, P. wickerhamii Tubaki et Soneda 1959, and P. zopfii Krfiger 1894. Although the individual Prototheca species differ morphologically, their accurate identification requires sugar and alcohol assimilation tests and/or immunofluorescence tests (3, 48). For rapid identification, API 20C yeast identification system is useful (39) (Table 1).
protothecosis (Table 2) (1, 2, 5-21, 23-33, 35-38, 40, 44-46, 49-57), and excluded a number of cases without evidence of infection (4, 43, 47). Analysis of the accepted cases showed the following results; (I) cutaneous/ subcutaneous protothecosis counted 24, synovial infection (protothecal bursitis/fasciitis/tenosynovitis) counted 21, and systemic protothecosis counted 5; 0I) the female to male ratio was 22:28; (//I) the age of the patients ranged from 5 to 80 (mean: 47); (IV) the 38 causal agents were identified as P. wickerhamii by the microbiological findings and/or by the immunofluorescent studies, 2 cases were caused by P. zopfii and agents from remaining 10 cases were described as Prototheca species; (V) the diseases was distributed worldwide (30 from North America, 13 from Asia including 6 from Japan, 2 each from Africa and Oceania, and 1 each from Europe, Central America, and South America).
Epidemiology
The first human case of cutaneous protothecosis was described by Davies et al. in 1964 (10). Most of 24 cases were affected on the exposed areas, especially on face and lower leg. The primary lesion is a small pink papule that occurs at the site of inoculation of etiologic agent. However, the traumatic injury could be unnoticed or it could have occurred years previously and forgotten subsequently. The papule gradually enlarges to form a erythematous plaque. The size of the lesions varies from coin size to more than half of the body surface (53). The lesion may be scaly or verrucous in appearance. Vesicular, herpetiform (15), ulcerative (33), multiple papular, and multiple nodular lesions have also been reported. Generally, the early lesions are asymptomatic or slightly itchy, and the histories of the disease are longer than several months, as in presented case. Most of the cases have systemic or localized immunocompromised conditions, such as systemic administration of corticosteroids (8 cases), diabetes (6 cases), renal failure (3 cases), and topical application of corticosteroids (8 cases).
Protothecosis is an infection of man and lower animals caused by members of the genus Prototheca. Although the disease is still rare, the number of the patient has been increasing worldwide. Over 100 cases of protothecosis have been reported in wide variety of wild and domestic animals, such as dogs, cows, cats, deer, and beavers. Bovine mastitis caused by P. zopfii is the most frequent and important form of the disase in animals. More than 50 cases of human protothecosis have been reported in the literature. We have critically evaluated these cases based on the clinical, histopathological, and microbiological descriptions presented by the original authors. An unclouded and strict criterion, i.e. Prototheca cells in the host's living tissue, was applied in the clinical entity. Cases without histopathological materials were reevaluated with the respective criteria. As a result of our critical evaluation, we have compiled 50 documented cases of human
Cutaneous/subcutaneous protothecosis
TABLE 1. - Identification of Prototheca species by API 20C system. Glucose
Glycerol
Galactose
Trehalose
Growth at 370 C
P. stagnora
+
+
+
-
-
P. wickerhamii
+
+
+/-
+
+
P. zopfii
+
+
-
-
+
(Padhye et al. (39), revised in part).
401
M a t s u d a T. a n d M a t s u m o t o T.
Eur. J. Epidemiol.
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Eur. J. Epidemiol. 0392-2990
Vol. 8, No. 3
JOURNAL
May 1992, p. 397-406
OF
EPIDEMIOLOGY
PROTOTHECOSIS: A REPORT OF TWO CASES IN JAPAN AND A REVIEW OF THE LITERATURE T. MATSUDA* and T. MATSUMOTO *.1 *Department o f Dermatology - FacuRy o f Medicine - Kyushu University - Fukuoka - Japan. **Department o f Dermatology - Toshiba General Hospital - 6-3-22 Higashi-oi - Shinagawa-ku Tokyo - Japan.
Key words: Prototheca - Protothecosis - Human cases Protothecosis is an emerging opportunistic infection caused by species belonging to the genus Prototheca. Two Japanese cases of protothecosis are documented with a critical review of the
literature. A current perspective concerning the microbiology and disease entity of protothecosis is described in detail.
INTRODUCTION
Protothecosis is a relatively rare infection caused by the species of the achlorophyllic genus Prototheca Krfiger, microorganisms morphologically similar to those of the genus Chlorella. The cases of protothecosis in human have been classified into the three clinical forms: 1) cutaneous and/or subcutaneous infection, 2)synovitis of olecranon bursa or other fibrous tissue, and 3) systemic infection. Recently, the disease has become very important both to physicians and microbiologists because of the increasing number of the reported cases, characteristic clinical features, and a distinct morphology of the etiologic agents in vitro as well as in vivo. The purposes of this presentation are to describe two Japanese cases of protothecosis, and to review and discuss of the literature of human protothecosis. Report of the cases Case 1.
An 80-year-old Japanese male patient noticed a spontaneous, asymptomatic red papule on his right 1 Corresponding author.
antecuboidal fossa in March 1987. He did not have any apparent antecedent traumatic history at the site of the lesion. He visited his neighbor physician, who diagnosed the lesion as chronic eczema. In spite of topical corticosteroid application for a couple of months, the lesion gradually spread, and he developed another extending plaque on his left arm. He consulted to the Dermatology Clinic, Kyushu University Hospital in August 1987. On physical examination, irregularly shaped, well demarcated, erythematous, infiltrated plaques are distributed symmetrically on the flexor surface of his arms. The surface of the lesions were slightly elevated and accompanied with vesicles, pustules, crusts, and fine scale (Fig. 1). No regional lymph nodes were palpable. He is a retired rice farmer in Kyushu island, the south-western area of Japan, and occasionally enters the rice paddy. Laboratory investigation revealed that he had an untreated diabetes and hyperglycemia. No immunological abnormality was detected. A skin biopsy specimen taken from a plaque on his right upper arm, stained with hematoxylin and eosin was histologically examined. In the dermis, there revealed a dense, granulomatous infiltration consisted mainly of lymphocytes, histiocytes, and scattered foreign body giant cells. Small, hyaline,
397
Matsuda T. and Matsumoto T.
Eur. J. Epidemiol.
Figure 1. - Clinical features of Case 1. Symmetrically distributed erythematous plaques on upper extremities.
round and yeast-like cells and large spherical cells were abundantly recognized within the multinuclear giant cells or in the interstitial tissue of the middle to reticular dermis. Further detailed observation of the microorganisms was performed with periodic acidSchiff staining. Small and single cells contained a large and single nucleus. Large cells were divided by nuclear and cytoplasmic cleveages, and appeared like wheels. Very large cells, containing 4-20 endospores, showed morula-like appearance (Fig. 2). Several pieces from a biopsy specimen were inoculated on Sabouraud dextrose agar plates and incubated at 25° C. From each of these, identical colonies were obtained in pure culture. The isolated organism yielded rapidly growing, smooth, moist, and creamy colonies. Microscopically, single cells were hyaline, globose to ovoid, and variable in size, measuring 3 to 12 lain in diameter. Some of the larger components were divided by cleavages, and endospores were contained within the others (Fig. 3). The mother cells were ruptured to release the spores, then each spore increased in size and eventually became mother cell. The isolate grew well at 37° C, and failed to hydrolyze urea. Assimilation test by the API 20 system showed positive results with galactose,
glucose, glycerol, and trehalose. On the basis of morphologic and physiologic characteristics, the isolate was identified as Prototheca wickerhamii Tubaki et Soneda. Immunohistopathologic study by anti-P. wickerhamii globulin, which was performed at the :Centers for Diseases Control's Division of Mycotic Diseases, also confirmed this identification. The patient was treated with oral administration of ketoconazole, a daily dose of 200 rag. Clinical effect appeared rapidly as the plaques discolored and the induration decreased. After 19 weeks of chemotherapy, each lesions healed leaving superficial scars. A biopsy specimen taken after 17 week ketaconazole therapy revealed no distinct microorganisms. The patient showed no recurrence during 3 year follow-up period, as of May 1991. Case 2.
A 13-year-old Japanese boy complained of general fatigue and palpitation in December 1987. He admitted to Hamanomachi Hospital in Fukuoka and was diagnosed as a patient with iron deficiency anemia due to gastrointestinal bleedings from unknown lesions. In October 1988, he developed a high fever
398
Vol. 8, 1992
Protothecosis from Japan
Figure 2. - Biopsy specimen showing spherical and wheel-like morphologies of etiologic agent accompanied by granulomatous inflammation (periodic acid-Schiff stain).
Figure 3. - Isolated microorganism showing various stages of its life cycle (wet preparation in lactophenol cotton blue).
399
Matsuda T. and Matsumoto T.
Eur. J. Epidemiol.
and severe general fatigue. Hepatosplenomegaly, liver abscesses, and enlarged abdominal lymph nodes were detected by means of body CT and upper abdominal echogram. On exploratory laparotomy, multiple, whitecolored, and small nodules were observed both in the submucosa of small intestine and on the liver surface. Mesenteric and ileocecal lymph nodes were swollen. Incisional biopsies were performed on each of these lesions. The white nodules in submucosa were revealed to be consisted mainly of Prototheca cells, which were surrounded by the dense inflammatory cellular infiltration (Fig. 4). In lymph nodes, abundant Prototheca cells were also recognized (Fig. 5). The isolation of the etiologic agent from stool materials were unsuccessful. However, the causative microorganism was later isolated from meningeal aspirate and identified as P. wickerhamii Tubaki et Soneda. Immunofluorescent study performed at the Centers for Disease Control's Division of Mycotic Disease also confirmed the identification. Intravenous amphotericin B injection for 11 weeks (total dose 1230 mg) was performed. On week 5
the patient developed headaches and double vision. Protothecal meningitis was suspected and intrathecal amphotericin B injection was also executed for 16 weeks (total dose 9.25 mg). The second laparotomy performed on week 23 revealed no significant lesion.
Figure 4. - Specimen form a submucosal nodule on intestine, consisted mainly of Prototheca cells (periodic acid-Schiff stain),
Figure 5. - Specimen from abdominal lymph node showing numerous Prototheca cells (periodic acid-Schiff stain).
Comment
Etiology The Prototheca species are achlorophyllic microorganisms morphologically similar to the green alga in the genus Chlorella. Prototheca zopfii, the type strain of the genus Prototheca, was originally isolated from the slime flux of trees by Krtiger in 1894 (22). Subsequent studies aroused a long lasting controversy concerning of the taxonomic position, alga or fungus, of this genus. Currently, the genus Prototheca is classified in alga on the basis of morphologic characteristics, life cycle, and reproduction system, common antigenic nature, and especially of the ultrastructural findings (34, 42). Morphologically, the Prototheca species grow rapidly on routine laboratory media without
400
Vol. 8, 1992
Protothecosis from Japan
cycloheximide. Colonies are soft, wet, yeast-like, and white to light tan in color. Microscopic examination reveals globose to ovoid hyaline cells measuring 1.3-16 lain in diameter. Each cell enlarges in size and makes nuclear division several times, followed by cleavage of the cytoplasm. Mature sporangium, containing 2 to 20 or more auto(endo) spores, ruptures and releases daughter cells. This reproductive cycle is repeated. Mycelium and conidia are absent. Species of the genus Prototheca are commonly isolated from various natural sources, such as slime flux of trees, soil, detritus, animal waste, and sewege (41). Four Prototheca species are currently recognized: P. salmonis Gentles et Bond 1977, P. stagnora W.B. Cooke 1968, P. wickerhamii Tubaki et Soneda 1959, and P. zopfii Krfiger 1894. Although the individual Prototheca species differ morphologically, their accurate identification requires sugar and alcohol assimilation tests and/or immunofluorescence tests (3, 48). For rapid identification, API 20C yeast identification system is useful (39) (Table 1).
protothecosis (Table 2) (1, 2, 5-21, 23-33, 35-38, 40, 44-46, 49-57), and excluded a number of cases without evidence of infection (4, 43, 47). Analysis of the accepted cases showed the following results; (I) cutaneous/ subcutaneous protothecosis counted 24, synovial infection (protothecal bursitis/fasciitis/tenosynovitis) counted 21, and systemic protothecosis counted 5; 0I) the female to male ratio was 22:28; (//I) the age of the patients ranged from 5 to 80 (mean: 47); (IV) the 38 causal agents were identified as P. wickerhamii by the microbiological findings and/or by the immunofluorescent studies, 2 cases were caused by P. zopfii and agents from remaining 10 cases were described as Prototheca species; (V) the diseases was distributed worldwide (30 from North America, 13 from Asia including 6 from Japan, 2 each from Africa and Oceania, and 1 each from Europe, Central America, and South America).
Epidemiology
The first human case of cutaneous protothecosis was described by Davies et al. in 1964 (10). Most of 24 cases were affected on the exposed areas, especially on face and lower leg. The primary lesion is a small pink papule that occurs at the site of inoculation of etiologic agent. However, the traumatic injury could be unnoticed or it could have occurred years previously and forgotten subsequently. The papule gradually enlarges to form a erythematous plaque. The size of the lesions varies from coin size to more than half of the body surface (53). The lesion may be scaly or verrucous in appearance. Vesicular, herpetiform (15), ulcerative (33), multiple papular, and multiple nodular lesions have also been reported. Generally, the early lesions are asymptomatic or slightly itchy, and the histories of the disease are longer than several months, as in presented case. Most of the cases have systemic or localized immunocompromised conditions, such as systemic administration of corticosteroids (8 cases), diabetes (6 cases), renal failure (3 cases), and topical application of corticosteroids (8 cases).
Protothecosis is an infection of man and lower animals caused by members of the genus Prototheca. Although the disease is still rare, the number of the patient has been increasing worldwide. Over 100 cases of protothecosis have been reported in wide variety of wild and domestic animals, such as dogs, cows, cats, deer, and beavers. Bovine mastitis caused by P. zopfii is the most frequent and important form of the disase in animals. More than 50 cases of human protothecosis have been reported in the literature. We have critically evaluated these cases based on the clinical, histopathological, and microbiological descriptions presented by the original authors. An unclouded and strict criterion, i.e. Prototheca cells in the host's living tissue, was applied in the clinical entity. Cases without histopathological materials were reevaluated with the respective criteria. As a result of our critical evaluation, we have compiled 50 documented cases of human
Cutaneous/subcutaneous protothecosis
TABLE 1. - Identification of Prototheca species by API 20C system. Glucose
Glycerol
Galactose
Trehalose
Growth at 370 C
P. stagnora
+
+
+
-
-
P. wickerhamii
+
+
+/-
+
+
P. zopfii
+
+
-
-
+
(Padhye et al. (39), revised in part).
401
M a t s u d a T. a n d M a t s u m o t o T.
Eur. J. Epidemiol.
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