Entomophthroramycosis

ENTOMOPHTHORAMYCOSIS

Abhijit Chaudhury

TAXONOMICAL CONSIDERATIONS

• Originally, fungi were part of the Plant Kingdom (Plantae), and subdivision Thallophyta.

• Three classes were recognised: Phycomycetes, Ascomycetes, Basidiomycetes (based on the morphologic similarities of sexual reproductive structures), and Deuteromycetes (Asexual reproduction).


• In 1969, Whittaker created the Kingdom Fungi.[Whittaker RH. New concepts of kingdoms of organisms. Evolutionary relations are better represented by new classifications than by the traditional two kingdoms. Science 1969; 163:150–60].

• The agents causing mucormycosis, entomophthoramycosis,and other fungi that produce coenocytic (aseptate) vegetative hyphae and sexual spores called ‘‘zygospores’’ or ‘‘oospores’’ were classified in the Phycomycetes group. [Emmons CW, Binford CH, Utz JP, Kwon-Chung KJ. Medical mycology.3rd ed. Philadelphia: Lea & Febiger, 1977:254–84].


• Because Phycomycetes comprised a miscellaneous assemblage of evolutionarily unrelated organisms, the class Phycomycetes was abolished [Whittaker, 1969], and the members of Phycomycetes were accommodated in a series of classes: Zygomycetes, Chytridiomycetes, Hypochytridiomycetes, Trichomycetes, and Oomycetes [Ainsworth GC. Introduction and keys to higher taxa. In: Ainsworth GC, Sparrow FK, Sussman AS, eds. The fungi. IVA. A taxonomic review with keys. New York: Academic Press, 1973:1–7].


• Further classification of the fungal kingdom on the basis of shared, derived characters delimited the kingdom to include only Chytridiomycota, Zygomycota, Ascomycota, and Basidiomycota, and this classification scheme has been universally accepted until a decade ago.

• The phylum Zygomycota contained Mucorales, Entomophthorales, and 8 other orders.


• Zygomycosis was originally described as a convenient and inclusive name for 2 clinicopathologically different diseases: Mucormycosis caused by members of Mucorales and Entomophthoramycosis caused by species in the order Entomophthorales of Zygomycota.


• Zygomycota underwent major taxonomic changes in 2007.

• Hibbet and others proposed to eliminate Zygomycota and the taxa conventionally placed in Zygomycota were distributed among the phylum Glomeromycota and 4 subphyla of uncertain placement.


• The Mucorales and Entomophthorales, which contain zoopathogenic fungi, and 2 other orders including Kickxellales and Zoopagales were nraised to the rank of subphyla: Mucoromycotina, Entomophthoromycotina, Kickxellomycotina, and Zoopagomycotina.

(Hibbett DS, Binder M, Bischoff JF, et al. A higher-level phylogenetic classification of the Fungi. Mycol Res 2007; 111:509–47).

Nomenclature of Disease

• Emmons proposed the name ‘‘phycomycoses’’ to provide a convenient and an inclusive term for mycoses caused by any one of the several species of Phycomycetes.

• ‘‘Phycomycosis’’ became widely accepted as a convenient disease name, irrespective of its diversity in clinical course and etiology .


• Clark, in 1968, however, supported use of the term ‘‘mucormycosis’’ for the diseases caused by species of Mucorales, to distinguish them from ‘‘subcutaneous phycomycosis’’ caused by fungi belonging to Entomophthorales.

• She also proposed the name ‘‘Entomophthoromycosis’’ for subcutaneous phycomycosis. (Clark BM. The epidemiology of phycomycosis. In: Wolstenholme GEW, Porter R, eds. Systemic mycoses. London: J & A Churchill, 1968:179–205)


• The names ‘‘mucormycosis’’ and ‘‘entomophthoramycosis’’ have been embedded in the medical literature for .50 years, and the time has come to replace ‘‘zygomycosis’’ with these 2 names. [ Kwon-Chung KJ. Taxonomy of Fungi Causing Mucormycosis and Entomophthoramycosis (Zygomycosis) and Nomenclature of the Disease: Molecular Mycologic Perspectives. Clinical Infectious Diseases 2012;54(S1):S8–15].


MUCORMYCOSIS• Worldwide• Produce numerous deciduous

asexual spores within the sporangium (sporangiospores).

• Zygospores of Mucorales are produced between2 opposed suspensors originating from different hyphae.

• Cause an acute angioinvasive infection primarily in immunocompromised individuals.

• Absent

Entomophthoramycosis• Tropical and subtropical • Produce a single conidium (with

no sporangium) on each conidiophore.

• Zygospores of Entomophthorales are produced by the union of 2 contiguous cells of a hypha.

• Produce chronic and subcutaneous infection mostly in immunocompetent individuals.

• Thick eosinophilic sleeves surround the fungal hyphae.

[ Kwon-Chung KJ. Taxonomy of Fungi Causing Mucormycosis and Entomophthoramycosis (Zygomycosis) and Nomenclature of the Disease: Molecular Mycologic Perspectives. Clinical Infectious Diseases 2012;54(S1):S8–15].

[ Kwon-Chung KJ.2012].

Historical Aspects

• The first well-documented case of disease caused by members of Mucorales was published in 1885 by the German pathologist Paltauf.

• It was a systemic infection with gastric and rhinocerebral involvement, which Paltauf described as ‘‘Mycosis Mucorina’.

[Paltauf A. Mycosis mucorina: ein Beitrag zur Kenntnis der menschilchen Fadenpiltzer-krankungen. Virchows Arch Pathol Anat 1885; 102:543–64].

Historical Aspects

• The disease name ‘‘mucormycosis’’ was subsequently used by the American pathologist R. D. Baker to denote a mycosis caused by certain members of Mucorales. [Baker RD. Mucormycosis, a new disease? JAMA 1957; 163:805–8].

• Entomophthoramycosis (Basidiobolomycosis) was first reported in 3 Indonesian children in 1956. [Kian Joe L, Njo-Injo TE, Pohan A, Van der Muelen H, Emmons CW. Basidiobolus ranarum as a cause of subcutaneous phycomycosis in Indonesia. AMA Arch Dermatol 1956; 74:378–83].

Historical Aspects

• Cases were subsequently reported from Brazil, India, Tropical Africa, SE Asian Countries.

• Indian isolates were classified as B. haptosporus. [Srinivasan M, Thirumalachar M. Basidiobolus species pathogenic for man. Sabouraudia 1965.; 4: 32-34].

• Later, the consensus was that B.haptosprus is identical to B. ranarum and the later name has priority.

Historical Aspects

• The first human case of Conidiobolomycosis was reported in 1965 in a patient from West Indies. [Bras G et al. A case of phycomycosis observed in Jamaica. Am J

Trop Med Hyg 1965; 14: 141-145]. • Systemic Conidiobolomycosis was reported in

1970 (Pericarditis).• Disseminated Conidiobolomycosis with death

was reported in 1984 caused by C. incongruus.

Human Aetiological Agents

• Basidiobolus ranarum• Conidiobolus coronatus• C. incongruus• Conidiobolus lamprauges

Epidemiology: A. Basidiobolus ranarum

• Reported mainly in tropical areas of Asia (India, Indonesia, and Myanmar), Africa (Uganda, Nigeria, Cameroon, Togo, Ivory Coast, Sudan, Senegal, Somalia, and Kenya), South America (mostly Brazil), North America (Mexico), and recently Australia. [Kwon-Chung K.J., Bennett J.E.: Entomophthoramycosis. In Medical Mycology. Philadelphia: Lea and Febiger, 1992].


• The fungus occurs in decaying vegetation, soil, and as a saprobe in the intestinal contents of various insectivorous reptiles (lizards, chameleon), amphibians (toads), and mammals (bats, kangaroos, and wallabies)[Kafor J.I., Testrake D., Mushinsky H.R.,et al. A Basidiobolus sp. and its association with reptiles and amphibians in Southern Florida . Sabouraudia: J Med Vet Mycol 1984; 22: 47]

• Infections caused by Basidiobolus ranarum are mainly diagnosed in children (80% under the age of 20 years) with a male/female ratio of 3:1.


• The portal of entry is believed to be the skin after insect bites, scratches, and minor cuts. This helps to explain the most common presentation in young children involving the thighs and buttocks. However, there is rarely a history of previous trauma.

[Elias J. Anaissie, Michael R. McGinnis, and Michael A. Pfaller Eds. Clinical Mycology ,2009; 2nd Ed. Chapter 12, 297-307; Elsevier]

Epidemiology: B. Conidiobolus coronatus

• Conidiobolus coronatus infections have been reported from tropical portions of Africa (mostly Cameroon and Nigeria, but also chad, Zaire, Kenya, Central African Republic, Guinea) and the Americas (Costa Rica, Caribbean islands, Columbia, Brazil). [Drouhet E., Ravisse P.: Entomophthoromycosis. In Borgers M., Hay R., Rinaldi M.G.(eds) Current Topics in Medical Mycology. Barcelona, Spain: J.R. Prous, 1993]


• The fungus is found in decaying wood, plant detritus, on insects, and in the gastrointestinal tract of lizards and toads.

• There are seasonal variations in the yield of C. coronatus from soil, a maximum being recorded in September and October, which suggests an influence of climate on spore survival, which may also help explain the geographic distribution of the infection. [Kwon Chung and Bennett, 1992]


• There is a male/female ratio of 10:1 and a predominance of the disease among young adults. Infection is rare among children. There is no known underlying predisposing factor for the infection. [Drouhet E., Ravisse P, 1993.]

• The spores are believed to enter the body by inhalation and then invade tissues through wounded nasal mucosa.

Clinical Manifestations

• Clinical entities are chronic, often indolent, and not life-threatening infections except in anecdotal cases of disseminated infections.

• Histologic features are identical, but clinical features differ.

Clinical Manifestations: Basidiobolomycosis

• Most cases seen in children.• The presenting feature is a single painless,

unilateral, well-circumscribed subcutaneous mass that usually affects the buttock or the thigh but can also be seen in the arm, the neck, the face or the trunk.

• The disease starts as a single nodule that progressively grows. The swelling is often described as woody and hard.

Clinical Manifestations: Basidiobolomycosis

• There is no ulceration and the mass is not adherent to deeper tissues, although involvement of muscle had been described.

• Enlargement of local lymph nodes is sometimes seen, with the fungus sometimes being cultured from the corresponding biopsy specimens.

• The lack of draining sinuses, the absence of adherence to underlying structures, and the lack of extension to bone make the differential diagnosis with mycetoma easy.

• Unusual localization includes gastrointestinal infection.

Clinical Manifestations: Conidiobolomycosis

• The infection starts in the nasal mucosa and progressively extends to adjacent areas bilaterally, including the nose, cheeks, upper lip, paranasal tissues, and pharynx.

• The edema affecting all the infected areas leads to significant distortion of the face.

• Apart from obvious changes in appearance, the patient may complain of nasal obstruction, rhinorrhea, and epistaxis.


• Invasion of the pharynx may cause dysphagia. • The lesion does not usually involve the bones• The evolution of the infection is slow over years.• There is no tendency for the mass to ulcerate or

become verrucous. • The mass is usually anchored to the dermis. • There is usually no fever and no biologic signs of

infection. Blood cell count and chemistry are normal


• Three cases of infections due to C. incongruus have been described so far.

• One occurred in an immunocompromised patient, in whom the initial pulmonary infection was rapidly fatal after spreading to the pericardium and heart.

• The two other cases occurred in a 15-month-old boy and a 20-year-old woman with no underlying disease.

• The infection initially involved the lungs and mediastinum with dissemination to adjacent tissues and eventually caused death of the young woman from massive hemoptysis; but the boy survived.

THERAPY

• Surgery: Surgical resection alone is not effective in managing infections caused by Basidiobolus or Conidiobolus spp. Cosmetic surgery can be proposed after prolonged antifungal therapy and sterilization of the lesion.

ANTIFUNGAL THERAPY

• Treatment is not well defined for entomophthoraceous fungi.

• Saturated potassium iodide (30 mg/kg/day) has long been the treatment of choice for chronic infections caused by Basidiobolus and Conidiobolus [Kwon-Chung and Bennett 1992, Drouhet and Revisse 1993].

ANTIFUNGAL THERAPY

• Since the discovery of azoles, patients have improved with, if not been cured by, ketoconazole or itraconazole.

• The efficacy of fluconazole ranges from complete cure, to partial improvement or failure.

• Amphotericin B is rarely prescribed for chronic infections [Fournier S., Dupont B., Begue P.,et al. Infection rhino-faciale Conidiobolus coronatus avec lyse osseuse et adenomegalie. Difficultes therapeutiques . J Mycol Med 1995; 5: 35]

LABORATORY DIAGNOSIS AND MYCOLOGICAL ASPECTS

A. DIRECT EXAMINATION: Punch biopsy material/ scrapings of nasal

mucosa mounted in KOH. Broad, non-septate or sparsely septate

hyphae with refractile walls and granular inclusions may be seen.

B. Histopathology

• Chronic inflammatory process can be seen with small abscesses surrounded by a granulomatous tissue reaction.

• A strong eosinophilic perihyphal reaction is often observed (Splendore–Hoeppli phenomenon) that is variable in size (2–6 μm). [Rippon J.W.: Zygomycosis. In Rippon J.W.(eds) Medical Mycology. The Pathogenic Fungi and the Pathogenic Actinomycetes. Philadelphia: WB Saunders, 1988]

• Broad irregular hyphae (4–30 μm) with thin walls and rare septation can be seen, singly or in clusters.

• There is no invasion of blood vessels or infarction of tissue,

Entomophthoromycosis caused by "Conidiobolus coronatus" - Haematoxylin and eosin (H&E) stained section of tissue showing broad sparsely septate hyphae surrounded by an eosinophilic sheath (Splenodore-Hoeppli phenomenon) typical of Entomophthoromycosis. (Courtesy Dr R. Garrison, V.A. Medical Centre, Kansas City, U.S.A.).

http://www.mycology.adelaide.edu.au/gallery/zygomycetes/

C. CULTURE

• Scrapings from nasal mucosa, biopsy material from the polyps, or material from skin biopsy are used as specimen.

• Biopsy specimen should be cultured immediately as B.ranarum dies quickly in tissues kept in a refrigerator.

• Tissue biopsy should be minced and not homogenized. Homogenization in a tissue grinder should be avoided, because it decreases culture yield by destroying hyphae.

• SDA with antibiotics can be used. Cycloheximide should not be used in SDA.

• Incubation at 250-300 C for 2-5 days.

Basidiobolus ranarum: Culture Characters and Morphology

• Colonies are yellowish to gray; thin, flat, glabrous, and waxy with many radial folds.

• The hyphae are wide (8-20µm) with occasional septa in young cultures, but become increasingly septate as sporulation proceeds.

• Sporulation become evident within 10 days with development of zygospores.


• Zygospores are smooth walled and are formed after the conjugation of two adjacent hyphal cells.

• A prominent beak is found attached to one side which is the remnant of conjugation tube.

• Conidia are formed when the apical portion of the conidiophore has enlarged and the globose conidium is blown out from the tip of this swelling.


• The swollen part becomes the subconidial vesicle.

• It also produces passively detached,elongate, adhesive conidia.

• Meristospore: The cytoplasm of some conidia cleaves into sporangiospores.

• A medium containing casein hydrolysate, low glucose, and glucosamine enhances sporulation.

ZYGOSPORES

p: primary conidia are forcibly discharged, with the remnant wall of ruptured conidiophores, s: secondary conidia, and a: ellongate conidia with an apical adhesive knob(capilliconidia) produced from a thin hypha.

(A, B, and C) Sporangiospores of B. ranarum showing cleavage formation to produce meristospores (black arrow), knob-like adhesive tip (white arrow), and ballistospores with

hyphal tag (white arrowhead). D) Thick-walled zygospores. (E) A zygospore with a beak.

Khan Z U et al. J. Clin. Microbiol. 2001;39:2360-2363

Conidiobolus coronatus: Culture Characters and Morphology

• Rapidly growing colony.• Creamy, glabrous, and waxy at first ; becomes

powdery when short, white aerial hyphae develop.

• Vegetative hyphae are frequently septate.• With age, colour of colony becomes buffy.• The inner side of plate or tube is covered by

white powdery material (forcibly discharged spores).

3 Days growth on PDA


• Conidiophores: 12-18x60-90 µm size originate from engorged hyphal segments.

• Conidiophores are phototrophic, and discharge conidia towards light source.

• Conidia are multinucleate, and globose with conspicuous basal papilla, which marks the former point of attachment to the conidiophore.


• The conidiophore releases the primary conidia up to 4 cm into the air using the evertion mechanism which is the characteristic feature of the Conidiobolus genus.

• The conidia also produces multiple, short, hair-like appendages called villi, which separates C.coronatus from C. incongruus.

Conidiobolus incongruus: Culture Characters and Morphology

• Colony similar to C.coronatus• Yellowish zygospores• No villous conidia• Primary conidia have sharply pointed papilla.

(A) CT scan of the midface: signs of sinusitis (*) and osteolyses of the medial part of the right orbit(→ (B) Hyphae with orthogonal branches in periodic acid-Schiff staining (magnification, ×600) in the biopsy specimens of the ethmoidal cells. (C to E) Micromorphology of Conidiobolus incongruus (lactophenol blue; magnification, ×1,000). (C and D) Wide vegetative mycelium with moderate septation. (D and E) Large single-celled primary conidia with pointed papillae. (F) Septate hyphae with orthogonal branches in the calcofluor white staining from the biopsy specimens of the right eye (postmortem; magnification, ×400). (G) Perivascular accumulation of fungal hyphae, with infiltration of the vessel wall and beginning infiltration of surrounding brain tissue in the frontal cortex (Grocott stain; magnification, ×200).

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Entomophthroramycosis