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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5575_Библиотеки_им_академика_М_И_Перельмана
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(b)
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(c)(a)
20 ˜m
20 ˜m
Fig. 11.1. Basidiobolus ranarum showing (a) culture with satellite colonies formed by germinating conidia ejected from
the primary colony, (b) conidia and a sporophore with a distinct swollen area just below the conidium (arrow), and
(c) thick-walled “beaked” zygospores.
DOI: 10.1079/9781800622340.0011
Descriptions of Medical Fungi 36
12 Beauveria bassiana (Balsamo)
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Vuillemin
Three species of Beauveria are recognised, two of which are well known parasites of insects. B. bassiana is
the most common species and is best known as the causal agent of the disastrous muscardine in silkworms.
rare human cases of keratitis have been reported (Lara Oya et al., 2016).
RG-1 organism
Morphological description: Colonies are usually slow growing, mostly not exceeding 2 cm in ten days at
o
20
C, downy, at first white but later often becoming yellow to pinkish with age. The genus Beauveria is
characterised by the sympodial development of single-celled conidia (ameroconidia) on a geniculate or
zig-zag rachis. Conidiogenous cells are flask-shaped, rachiform, proliferating sympodially and are often
aggregated into sporodochia or synnemata. Conidia are hyaline and globose or ovoid in shape (Fig. 12.1).
20 ˜m
Fig. 12.1. Beauveria bassiana showing sympodial development of conidia on a geniculate or zig-zag rachis.
© CAB International 2023. Descriptions of Medical Fungi (eds S. Kidd, C.Halliday and D. Ellis)
37
Key features: Sympodial development of single-celled conidia on a geniculate or zig-zag rachis emanating
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from a flask-shaped conidiophore.
Molecular identification: Specific primers were developed by Hegedus and Khachatourians (1996). Full
phylogeny of the genus was provided by Rehner and Buckley (2005). Biogeography of molecular types was
elaborated by Ghikas et al. (2010).
MALDI-ToF MS: Cassagne et al. (2011) published a standardised procedure for mould identification in the
clinical laboratory.
References: de Hoog (1972); McGinnis (1980); Domsch et al. (2007); de Hoog et al. (2015).
DOI: 10.1079/9781800622340.0012
Descriptions of Medical Fungi 38
The genus Bipolaris contains about 47 species, which are mostly subtropical and tropical plant parasites.
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Bipolaris cynodontis and B. papendorfii have been isolated from clinical samples (da Cunha et al., 2012a).
However recent phylogenetic studies have transferred all the well-documented human pathogens, notably
B.australiensis, B. hawaiiensis and B. spicifera to the genus Curvularia (Manamgoda et al., 2012). Other
reported clinical isolates include Curvularia micropus (formerly Bipolaris micropus) and Cochliobolus
setariae (formerly Bipolaris setariae).
RG-1 organisms
Morphological description: Colonies are moderately fast growing, effuse, grey to blackish-brown, suede-like
to floccose with a black reverse. Microscopic morphology shows sympodial development of pale brown
pigmented, distoseptate conidia on a geniculate or zig-zag rachis. Conidia mostly curved, canoe-shaped,
fusoid or obclavate, rarely straight, 2-14 distoseptate (usually more than 6), germinating only from the ends
(bipolar).
Key features: Darkly pigmented mould producing sympodial, distoseptate, pale brown, straight, fusiform to
ellipsoidal conidia, which are rounded at both ends.
Comments: The genera Drechslera, Bipolaris, Curvularia and Exserohilum are all closely related and mor-
phological differentiation of the genera relies upon a combination of characters including conidial shape, the
presence or absence of a protruding hilum, the contour of the basal portion of the conidium and its hilum,
the point at which the germ tube originates from the basal cell and, to a lesser degree, the sequence and location of the first three conidial septa. However, Manamgoda et al. (2012) reported no clear morphological
distinction between the genera Bipolaris and Curvularia and some species have intermediate morphologies
between the two genera. These authors recommend using a combined ITS and GAPDH sequence analysis for
definitive identification of species (Manamgoda et al., 2012, 2014).
Molecular identification: ITS sequencing may be used to identify clinical species (da Cunha et al., 2012a).
GAPDH (Glycerol-3-phosphate dehydrogenase) has been determined to be the optimal phylogenetic marker
of Bipolaris species (Manamgoda et al., 2012, 2014).
References: Ellis (1971, 1976); Alcorn (1983); McGinnis et al. (1986b); Sivanesan (1987); Rippon (1988);
Domsch et al. (2007); de Hoog et al. (2015).
DOI: 10.1079/9781800622340.0013
© CAB International 2023. Descriptions of Medical Fungi (eds S. Kidd, C.Halliday and D. Ellis)
39
14 Blastomyces Gilchrist & Stokes
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Blastomyces contains a number of pathogenic species, most notably Blastomyces dermatitidis and
Blastomyces gilchristii, which are morphologically identical but distinguishable by sequence analysis of the
ITS region (Brown et al., 2013). These are associated with soil and decaying organic matter such as leaves
and wood in North America. Blastomyces percursus and B. emzantsi have been identified from a culture
repository in South Africa (Dukik et al., 2017; Jiang et al., 2018; Maphanga et al., 2020). In addition,
Blastomyces helices and B. parvus have been transferred to this genus from Emmonsia (Peterson and Sigler,
1998; Jiang et al., 2018). These are the causative agents of blastomycosis a chronic granulomatous and suppurative disease, having a primary pulmonary stage that is frequently followed by dissemination to other
body sites, typically the skin and bone.
10 ˜m
Fig. 14.1. Blastomyces dermatitidis culture and microscopy showing one-celled, smooth-walled conidia borne on
short lateral or terminal hyphal branches (photographs courtesy of Jon Biehle, Department of Pathology, Creighton
University, Omaha, Nebraska).
40
© CAB International 2023.
Descriptions of Medical Fungi (eds S. Kidd, C.Halliday and D. Ellis)
WARNING: RG-3 organism
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Cultures of Blastomyces species represent a biohazard to laboratory personnel and must be handled in a
Class II Biological safety cabinet (BSCII).
14.1. Blastomyces dermatitidis Gilchrist & Stokes
Morphological description: Colonies grown at 25oC are variable in morphology and rate of growth. They
may grow rapidly, producing a fluffy white mycelium or slowly as glabrous, tan, non-sporulating colonies.
Growth and sporulation may be enhanced by yeast extract. Most strains become pleomorphic with age.
Microscopically, hyaline, ovoid to pyriform, one-celled, smooth-walled conidia (2-10 μm in diameter) of the
Chrysosporium type, are borne on short lateral or terminal hyphal branches (Fig. 14.1).
Blastomyces species are dimorphic fungi and colonies on blood agar at 37
glabrous and yeast-like. Microscopically, the organism produces the characteristic yeast phase as seen in tissue pathology.
Comment: In the past, conversion from the mould to the yeast form was necessary to positively identify this
dimorphic pathogen from species of Chrysosporium or Sepedonium. However, identification by molecular
methods is preferred to minimise manipulation of the fungus.
Histopathology: Blastomyces dermatitidis tissue sections show large, thick-walled, broad-based, unipolar
budding yeast-like cells, which may vary in size from 8-15 μm, with some larger forms up to 30 μm in diameter. Tissue sections need to be stained by Grocott-Gomori’s methenamine silver method to clearly see the
yeast-like cells, which are often difficult to observe in haematoxylin and eosin preparations (Fig. 14.2).
(a) (b)
o
C are wrinkled and folded,
20 ˜m
Fig. 14.2. Blastomyces dermatitidis showing large, broad-base, unipolar budding yeast-like cells (arrows) in
(a) haematoxylin and eosin-stained section of lung tissue and (b) calcofluor white stain of the yeast phase grown
at 36oC (photographs courtesy of Jon Biehle, Department of Pathology, Creighton University, Omaha, Nebraska).
Descriptions of Medical Fungi 41
10 ˜m
Molecular identification: Several conventional PCR assays have been developed for the identification of
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B.dermatitidis from clinical specimens (Bialek et al., 2003) and soil (Burgess et al., 2006). Sidamonidze etal.
(2012) developed a real-time PCR targeting the BAD1 (formerly known as WI-1) gene for the identification
of B. dermatitidis in culture and tissue and Morjaria et al. (2015) used ITS and D1/D2 sequencing for
identification from paraffin embedded tissue.
Key features: Clinical history, tissue pathology, culture identification by positive exoantigen test.
Antifungal susceptibility: (Table 14.1).
Table 14.1. Blastomyces dermatitidis limited data available (Sugar and Liu, 1996; Espinel-Ingroff et al., 2001; Espinel-Ingroff,
2003; Gonzalez et al., 2005; Sabatelli et al., 2006); MIC μg/mL. Antifungal susceptibility testing is not recommended.
Antifungal Range MIC
FLU 0.125-64 4-16 AmB 0.03-1 0.5
ITRA 0.03->16 0.125-2 VORI 0.03-16 0.25
POSA 0.03-2 0.125
90
Antifungal Range MIC
90
References: Chandler et al. (1980); McGinnis (1980); Kaufman and Standard (1987); Rippon (1988); Jiang
et al. (2018).
DOI: 10.1079/9781800622340.0014
Descriptions of Medical Fungi 42
15 Candida Berkhout
(a) (b)
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The genus Candida is characterised by globose to elongate yeast-like cells or blastoconidia that reproduce by
narrow-based multilateral budding. Pseudohyphae and occasionally true hyphae may also be present. Colony
pigmentation is usually absent. Ballistoconidia are not formed. Arthroconidia may be formed, but not
extensively. Sexual reproduction is absent. Glucose may be fermented. Nitrate may be assimilated. Starch-like
compounds are not produced. The diazonium blue B reaction is negative.
The genus is highly polyphyletic, as it comprises mitosporic species that are devoid of special distinguishing
features (Lachance et al., 2011). Accordingly, several taxonomic rearrangements have been made and many
well-known Candida species have been moved to other genera, notably Pichia kudriavzevii (formerly
Candida krusei), Meyerozyma guilliermondii (formerly Candida guilliermondii), Clavispora lusitaniae (formerly Candida lusitaniae), Kluyveromyces marxianus (formerly Candida kefyr), Diutina catenulata (formerly
Candida catenulata), Diutina rugosa (formerly Candida rugosa) and Wickerhamomyces anomalus (formerly
Candida pelliculosa). Candida glabrata and C. parapsilosis are recognised as species complexes (Alcoba-
Florez et al., 2005; Tavanti et al., 2005; Correia et al., 2006).
Several species may be aetiological agents, most commonly Candida albicans, followed by C. glabrata,
C. parapsilosis, C. tropicalis and Pichia kudriavzevii. Altogether, these five species account for >95% of
human infections. However, a number of other species may also be isolated. All are ubiquitous and occur
naturally on humans.
Morphological identification: Ensure that you start with a fresh growing pure culture, streak for single
colony isolation if necessary (Fig. 15.1) (Kurtzman et al., 2011).
Fig. 15.1. Candida albicans showing (a) typical cream coloured, smooth-surfaced, waxy colonies and (b) narrow
based budding spherical to ovoid blastoconidia.
© CAB International 2023. Descriptions of Medical Fungi (eds S. Kidd, C.Halliday and D. Ellis)
10 ˜m
43
Chromogenic agars: Are now being used for primary isolation and for the detection of mixed flora, especially
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from non-sterile sites. Depending on the brand of chromogenic media presumptive identification of C. albicans, C. tropicalis, C. auris and P. kudriavzevii is possible. CHROMagar™ Candida (and Candida Plus)
(CHROMagar, Paris, France) is particularly useful for the detection of mixed infections (Fig. 15.2).
Fig. 15.2. CHROMagar™ Candida plate showing chromogenic colour change for Candida albicans (green),
C.tropicalis (blue), C. parapsilosis (white) and C. glabrata (mauve).
Germ tube test: A rapid screening test for Candida albicans and Candida dubliniensis. 0.5 mL of serum,
containing 0.5% glucose, is lightly inoculated with the test organism and incubated at 35
o
C for 2-3 hours.
On microscopy, the production of germ tubes by the cells is presumptive for Candida albicans and Candida
dubliniensis (Fig. 15.3).
Dalmau plate culture: To set up a yeast morphology plate, dip a flamed sterilised straight wire into a culture
to make a light inoculum and then lightly scratch the wire into the surface of a cornmeal/Tween 80, rice/
Tween 80 or yeast morphology agar plate, then place a flamed coverslip onto the agar surface covering the
scratches. Dalmau morphology plates are examined in situ directly under the lower power of a microscope
for the presence of pseudohyphae which may take up to 4-5 days at 26
o
C to develop. Candida albicans also
produces characteristic large, round, terminal, thick-walled vesicles (chlamydospores). For best results a light
inoculum should be scratched into the agar surface using a wire (Fig. 15.4).
Descriptions of Medical Fungi 44
10 ˜m
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Fig. 15.3. Candida albicans showing production of germ tubes.
(a) (b)
5 mm
Fig. 15.4. Candida albicans Dalmau plate culture showing (a) colonies growing out from scratches on the surface of a
cornmeal/Tween 80 agar plate, and (b) the production of large round, thick-walled chlamydospores. Note: A coverslip
has been placed onto the agar surface covering the scratches.
10 ˜m
Physiological and biochemical tests: Fermentation and assimilation studies should be performed based on
those used at the Westerdijk Biodiversity Institute (formerly known as the Centraalbureau voor
Schimmelcultures) (Kurtzman et al., 2011). Several yeast identification systems are commercially available,
for example the API 20C AUX (bioMerieux, Marcy-l’Etoile, France, ID 32C (bioMerieux), YT
MicroPlate (Biolog, Hayward, California), AuxaColor 2 (Bio-Rad, Hercules, California) and Vitek 2 ID-YST
(bioMerieux). However, they can only be used to identify those species in their respective databases and may
misidentify many uncommon or new yeasts, including Candida auris. Other supplementary tests include
growth at 37
Descriptions of Medical Fungi 45
o
C, cycloheximide resistance and hydrolysis of urea.
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