Pathological Findings
Prototheca is a large nonbudding cell readily seen in tissue. It is spheroid, ovoid, or elliptical with a prominent wall, and the round cell (theca) contains several thick-walled autospores (
108,
120). No budding is found.
Prototheca species are not easily apparent in hematoxylin- or eosin-stained smears but stain well with Gridley fungus stain, Grocott's modification of Gomori methenamine silver, or PAS, with or without diastase (Fig.
2) (
64,
75). The lack of characteristic endospores causes
Prototheca to resemble nonsporulating cells of
Blastomyces dermatitidis,
Cryptococcus neoformans,
Paracoccidioides brasiliensis, and some stages of
Coccidioides immitis,
Pneumocystis jiroveci,
Rhinosporidium seeberi, and the agent causing chromomycosis (
8,
38,
88,
114,
139,
140,
149,
162); thus, diagnosis of
Prototheca species infection by histopathology can be difficult. The size of the sporangia is helpful in distinguishing
Prototheca from other fungi. The sporangia of
Coccidioides are 10 to 100 times larger than those of
Prototheca species, and the individual endospores of
Coccidioides are smaller.
In tissue,
Prototheca organisms may appear morphologically similar to green algae (
30,
33,
131). Light microscopy revealed not only similarities in size, shape, and mode of reproduction but also a striking difference between the
Prototheca organisms and green algae. Unlike
Prototheca species, the green algae contained abundant cytoplasmic starch granules that were PAS negative following diastase digestion. PAS is particularly useful for differentiating the green algae from
Prototheca cells in tissue (
26,
58). Also, electron microscopy showed chloroplasts to be absent from
Prototheca spp.
Histopathologic studies describe a variety of host tissue responses, ranging from severe granulomatous necrosis to a total absence of inflammatory changes (
142). The histologic characteristics of cutaneous/subcutaneous and postoperative lesions have been variously described as granulomatous inflammation with necrosis; giant cells; a mixed infiltrate with plasma cells, lymphocytes, and histiocytes; hyperkeratosis; focal parakeratosis; pseudoepithelialization; hyperplastic lymphoid tissue; a dense chronic inflammatory cell infiltrate; and the presence of abundant organisms (
56,
91,
107,
109). Organisms are usually in the mid- to papillary dermis, although some involvement in the epidermis was reported earlier (
27,
162).
The histologic features of olecranon bursitis lesions consist of granulomatous inflammation with giant cells, epithelioid cells, lymphocytes, and plasma cells as well as organisms in tissue (
52,
56,
61). In dissemination, tissues show significant eosinophilia and fibrosis in the gall bladder, duodenum, and hepatic portal areas (
24,
25,
56).
Microbiological Tests
Diagnostic procedures for the identification of
Prototheca spp. should include the evaluation of characteristic micromorphology and specific assimilation patterns (
105,
114). Identification should not rely on the assimilation pattern alone, as
Prototheca sp. profiles can be identical to those of other yeasts.
The failure to isolate
Prototheca species may be explained by the fact that they are readily overgrown by bacteria and fungi when culture is attempted from contaminated sources.
Prototheca species have simple nutritional requirements and grow readily on a variety of synthetic media (
143). Yet many commonly used culture media are unsatisfactory and contain unsuitable nutrients or inhibitors (
118), such as cycloheximide, which is present in many selective fungal media (
18). Other media that may be useful include beef infusion broth, blood agar, and brain heart infusion agar (
6,
73). Pore (
118) suggested
Prototheca isolation medium for selective cultivation. The combination of flucytosine and potassium hydrogen phthalate inhibits most bacteria and fungi.
Prototheca isolation medium allows
Prototheca isolation from densely contaminated sources, such as sewage, soil, or stream water. Incubation at 30°C for 72 h is adequate for most
Prototheca species, while some slow-growing strains require incubation at 25°C for up to 7 days. Growth is optimized between 25 and 37°C, and organisms usually proliferate within 48 h as soft, wet, yeast-like, white-to-light-tan colonies. The organism can be either aerobic or microaerophilic (
121).
P. wickerhamii and
P. zopfii can be distinguished from
P. stagnora, which grows only at 30°C (
85).
Round-oval
Prototheca organisms with endospores can be unambiguously identified in native specimens (
148). A wet mount of the culture material may be stained with lactophenol cotton blue or calcofluor white to reveal the characteristic endosporulating sporangia (the so-called morula form) (Fig.
2).
P. wickerhamii and
P. zopfii differ in that
P. wickerhamii tends to form symmetrical morula forms, whereas
P. zopfii exhibits more random internal segmentation (
121).
The colony morphology of
Prototheca zopfii Krüger 1894 on Sabouraud glucose agar at 25°C is dull white and yeast-like in consistency (Fig.
2). The cells are variable in size and shape, being 8.1 to 24 μm by 10.8 to 26.9 μm. The autospores are spherical and 9 to 11 μm in diameter (Table
3). Morphology varies depending on the medium employed for growth (
3,
72,
120).
Colony morphology for
P. wickerhamii Tubaki and Soneda 1959 on Sabouraud glucose agar at 25°C is moist and cream-colored. Growth is optimal at 30°C, and the cells are similar to those of
P. zopfii in shape but are somewhat smaller. The cells vary from 8.1 to 13.4 μm by 10.8 to 16.1 μm when grown on glucose-containing media. The autospores are smaller (4 to 5 μm in diameter) and more numerous (up to 50 per theca) (Table
3) (
3,
72,
120).
P. stagnora Cooke 1968 is mucoid, owing to the capsular material it produces (Table
3). Unlike
P. zopfii and
P. wickerhamii, it does not grow well at 37°C (
3,
72,
120).
Prototheca organisms can be identified to the species level by using the API strip series (bioMérieux, Marcy l'Etoile, Paris, France) applicable for yeasts (
105), the Vitek yeast identification database (bioMérieux, Marcy l'Etoile, Paris, France) (
42), the Vitek 2 test (bioMérieux, Marcy l'Etoile, Paris) (
78), and the RapidID Yeast Plus test (Remel, Santa Fe, NM) (
44,
78). The API 20C clinical yeast system is a ready-to-use micromethod permitting the performance of 19 assimilation tests for the identification of most clinically significant yeast species. Biochemical reactions are complete after 72 h of incubation. The API 20C system provides an opportunity to determine the assimilation patterns of
P. stagnora,
P. wickerhamii, and
P. zopfii (
92,
105). Yet both the API 20C and Vitek databases include only
P. wickerhamii for identification.
The API 50 system is also a ready-to-use micromethod that permits the assimilation patterns of 50 carbohydrates to be studied (
105). The system is not available commercially. Pal et al. (
106) described a new staining solution named “PHOL” for studying the morphology of clinical and environmental isolates of fungi and
Prototheca species. The solution has shown a good ability to stain isolates of fungi and
Prototheca and has the potential to stain the young as well as old isolates. Urease activity can be determined on Christensen urea agar at 30°C for 7 days, as
Prototheca species fail to hydrolyze urea (
105). For this examination, isolates of
Cryptococcus albidus and
Candida albicans should be used simultaneously as positive and negative controls, respectively.
Casal et al. (
21) showed colonies of
Candida parapsilosis and
Prototheca spp. growing on CHROMagar
Candida medium (CHROMagar Company, Paris, France) that were similar in color (cream) and texture after 48 to 72 h of growth, with
Prototheca colonies being slightly smaller than those of
C. parapsilosis. Given the increasing incidence of
C. parapsilosis in clinical processes and the possible occurrence of
Prototheca species in clinical samples, caution is warranted in using this medium. Also, CHROMagar
Candida medium allows no differentiation between
P. wickerhamii,
P. zopfii, and
P. stagnora, as all produce similar colonies.
Arnold and Ahearn (
3) developed a method for identification of the
Prototheca species, using carbohydrate and alcohol assimilation tests with the application of sucrose, trehalose, lactose, inositol,
n-propanol, and xylose as carbon sources. This auxanographic method is reliable but time-consuming. It may take up to 2 weeks to make a definitive identification. The absence of growth on trehalose is a main diagnostic feature for differentiation between
P. wickerhamii and
P. zopfii (Table
3).
The fluorescent antibody technique (
56,
143) is a helpful tool for detection of
Prototheca organisms at the genus level.
An aggregation test distinguishes the
Prototheca genus from various types of the
Candida genus (
95) but is not available for commercial use. FTIR has been reported to be a suitable and efficient method for distinguishing and characterizing human-pathogenic yeasts and animal-pathogenic algae. Also, FTIR allows differentiation between
P. zopfii and
P. wickerhamii (
132). However, for routine diagnosis, more conventional methods, such as cultivation and microscopic examination, seem to be sufficient (
148). The detection of antibodies proved to be useful in the diagnosis of clinical and subclinical protothecal mastitis (
11,
128) in cattle. Yet a serologic survey of
P. wickerhamii in wastewater workers yielded negative results (
31).
Another simple and rapid method for differentiating
Prototheca species from
Candida was described by Casal and Gutierrez (
17,
18). The algaecide ribostamycin inhibits growth of
Prototheca species but not that of
Candida species or other yeasts at 60 μg ribostamycin per lamella. Also, clotrimazole is useful in separating
P. wickerhamii from
P. zopfii, using 50-μg clotrimazole disks.
P. zopfii tested in a study was resistant, and
P. wickerhamii was susceptible (
20). Susceptibility to neomycin is helpful in differentiating
P. wickerhamii and
P. zopfii from
P. stagnora (
16).