Emergomyces: The global rise of new dimorphic fungal pathogens

1 Division of Infectious Diseases, Department of Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada, 2 National Institute for Communicable Diseases, a Division of the National Health Laboratory Service, Johannesburg, Gauteng, South Africa, 3 Department of Clinical Microbiology and Infectious Diseases, University of the Witwatersrand, Johannesburg, Gauteng, South Africa, 4 Department of Biological Sciences, Faculty of Sciences, University of Alberta, Edmonton, Alberta, Canada, 5 Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands, 6 Department of Dermatology, The Affiliated Hospital, Guizhou Medical University, Guiyang, China, 7 University of the Free State, Bloemfontein, Free State, South Africa, 8 Department of Microbiology, Stellenbosch University, Stellenbosch, Western Cape, South Africa, 9 Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, Western Cape, South Africa, 10 Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America, 11 Center of Expertise in Mycology of RadboudUMC/ Canisius Wilhelmina Hospital, Nijmegen, The Netherlands, 12 Global Health Institute, University of Antwerp, Antwerp, Belgium, 13 Clinical Sciences Unit, Institute of Tropical Medicine, Antwerp, Belgium, 14 Department of Medicine, University of Cape Town, Cape Town, Western Cape, South Africa


Origin story and current status
In 1994, an Italian woman with advanced HIV disease and widespread cutaneous lesions was diagnosed with an atypical disseminated mycosis. A biopsy of skin tissue demonstrated small budding yeasts, resembling those of Histoplasma capsulatum, in addition to larger pleomorphic cells. Moreover, the fungus that grew in culture had a dissimilar microscopic appearance [1,2]. The conidia were arranged in complex "florets" on slightly swollen stalks reminiscent of Emmonsia crescens or Ea. parva, fungi for which the thermodependent phase is characterized by swollen, thick-walled, nonreplicating cells called adiaspores, usually observed in the lungs of small terrestrial mammals in a disease called adiaspiromycosis [3]. Genetic analysis suggested the clinical isolate was related to Ea. crescens, and the fungus was therefore described in 1998 as Ea. pasteuriana [2].
In 2013, following the introduction of molecular identification procedures in some South African laboratories, a dimorphic fungus most closely related to Ea. pasteuriana was found to be the cause of a disseminated mycosis in patients with advanced HIV disease [4]. Since then, infection with this fungus has been established to comprise the most frequently diagnosed dimorphic mycosis in South Africa [5][6][7]. In light of these reports, global collections were reexamined for dimorphic Emmonsia-like fungi. Archived isolates were examined  [8,9]. Five species are now described within Emergomyces [8,9], and cases of disease have been reported globally (Fig 1). Es. pasteurianus, the type species, has been reported from Europe (including from Italy [1], Spain [10], France [ex-Georgia] [11], and the Netherlands [12]), Asia (China [13,14] and India [ex-Nepal] [15]) and Africa (Uganda [ex-Rwanda] [16] and South Africa [9]). Es. africanus has been reported from South Africa and Lesotho [4,9]. Es. canadensis has been reported from Canada (Saskatchewan) and the United States (Colorado and New Mexico) [17]. Es. orientalis has been reported from China [18], and Es. europaeus has been reported once from Germany [19]. The earliest known isolate among these Emergomyces species was from 1992 [17]. It remains unclear if these fungi have truly emerged or whether they are only now recognized because of an increase in the number of susceptible hosts, improved microbiology capacity, and/or the widespread adoption of molecular identification techniques in clinical and research laboratories.

Ecology
As with other systemic dimorphic fungi, infection is presumed to occur from inhalation of conidia, followed by a temperature-dependent transformation to a yeast-like phase that is capable of causing disease and of extrapulmonary dissemination (Fig 2).
Studies in search of an environmental reservoir for Emergomyces species have focused on Es. africanus in South Africa due to the large number of cases there [20][21][22]. Es. africanus DNA was detected in 30% of soil samples tested in the Western Cape Province, including from a wide range of soil habitats, such as soils associated with human activities like agriculture and horticulture and soils associated with plants endemic to the Cape Floral Kingdom. However, to date, the fungus has not been successfully isolated in culture from the environment, even with passage of soils through mice [20]. Es. africanus DNA was also detected in 10% of air samples collected in an urban location in the Cape Town metropole [21]. Naturally occurring infections of animals have not been demonstrated [22].

Emergomycosis
Disease caused by Emergomyces infection has been called emergomycosis (formerly disseminated emmonsiosis), and similarities have been noted among cases caused by different species. In general, patients with reported emergomycosis have been immunocompromised, including with HIV infection, solid organ transplantation, hematological malignancies, and immunosuppressant use [23]. Most patients with Es. africanus infection have had cutaneous lesions, which most commonly appear as papules, plaques, nodules, or ulcers, typically with widespread distributions [4,6]. Pulmonary disease is also common: 86% of patients had abnormal chest X-rays in a series from South Africa [6]. Chest radiograph abnormalities have included diffuse reticulonodular disease, consolidation, effusions, and/or lymphadenopathy [6]. Other sites of disease that have been reported include the gastrointestinal tract, liver, lymph nodes, and bone marrow [6]. Limited pulmonary disease has rarely been described, observed in the sole reported case of disease caused by Es. europaeus [19]. For other species, all reported cases have involved disseminated disease. This may reflect a diagnostic bias in clinical practice due to late disease presentation and limited access to more invasive (e.g., bronchoscopic) pulmonary sampling in resource limited settings or reporting bias in the medical literature.
The diagnosis of emergomycosis can be made by biopsy of affected tissue for histopathology and fungal culture. Histopathology findings include small (2-5-μm) yeasts with narrow-based budding, best seen with fungal stains [4]. The findings are insufficiently distinct from H. capsulatum to allow definitive identification from histopathological appearance alone [5]. The diagnosis can be confirmed by culture of Emergomyces species from clinical samples. Where fungal cultures are negative (or omitted), PCR of fresh, affected tissue using amplification and sequencing of the internal transcribed spacer (ITS) can establish the correct diagnosis [6,9].
Emergomyces species grow readily on standard fungal media (e.g., Sabouraud agar, malt extract agar, or potato dextrose agar), incubated at 24-30˚C. Colonies are yellowish white to tan, initially glabrous, becoming powdery, slightly raised, and furrowed, and reach diameters of 2.5 to 3.5 cm in 3 weeks. Microscopically, Emergomyces spp. are characterized in the mold phase by slender conidiophores that arise from hyphae at right angles and form "florets" of short secondary conidiophores bearing single small subspherical conidia (see Fig 2, step 1 [inset]). Conversion from the mold to the yeast phase occurs readily when colonies are streaked onto potato dextrose agar or malt extract agar and incubated at 35˚C [8, 9, 17].
Clinical microbiologists should be aware that Emergomyces species can cross react with a commercial DNA probe for B. dermatitidis [17]. There are neither sensitive nor specific serological tests nor biomarkers for the diagnosis of emergomycosis, though cross-reactivity can be observed with tests for related fungal infections [5,6]. Emergomyces species may cross react with Histoplasma urinary antigen tests [5, 6], but a negative test cannot exclude the diagnosis: in a series of 10 patients with culture-proven emergomycosis, only 3 tested positive by Histoplasma urinary antigen test [5].
Current treatment recommendations for emergomycosis are based on observational studies and expert opinion and are the same as those for patients with histoplasmosis [6,23]. Initially, immunocompromised patients with emergomycosis should be treated with amphotericin B for 10-14 days; where available, liposomal formulations are preferred over deoxycholate because of a more favorable toxicity profile. Thereafter, patients should be treated with itraconazole or another newer azole for 12 months pending immune reconstitution. Fluconazole should be avoided because high minimum inhibitory concentrations have been observed [7, 17,24]. Among HIV-infected persons with emergomycosis who are antiretroviral therapy (ART)-naive (or on a failing ART regimen), the optimal timing of ART initiation (or modification) has not been established.

Pathogenesis
There remains much to learn about the virulence factors of these fungi and pathogenesis involved in infection. Known virulence factor genes of dimorphic fungi are conserved in Es. africanus and Es. pasteurianus [25]. The role of these in Emergomyces pathogenesis still needs to be evaluated. Strains of Es. africanus, Es. europaeus, and Es. pasteurianus (but not Es. canadensis or Es. orientalis) express urease [8, 26], a known virulence factor for some pathogenic fungi like Cryptococcus neoformans and C. gattii. Experimental infections have demonstrated susceptibility of golden hamsters and mice [20,27]. Schwartz and colleagues found that intraperitoneal inoculations with Es. africanus were fatal to wild-type mice at doses of 10 6 conidia, whereas lower doses did not cause disease (although the organism could still be cultured from their livers and spleens with inoculae as low as 10 2 conidia) [20]. Moreover, C57BL/6 mice were more susceptible to disease than BALB/c mice [20]. Further work is underway to understand the pathogenesis of disease and the immunology of infection.

Future directions
There are many unresolved questions about Emergomyces. The true geographic range of Emergomyces species remains speculative, given the sporadic reports of disease from areas with limited mycological diagnostic capacity. For example, a case of emergomycosis caused by Es. pasteurianus in Uganda was diagnosed after a visiting doctor returned to Germany with a skin biopsy specimen that was collected from a Ugandan patient with a disseminated mycosis [16]. There, the diagnosis was made by nucleic acid amplification and sequencing, technology lacking in most African settings. It is presumed that this case and those diagnosed in South Africa represent the "ears of the hippo" on the African continent and that many other cases there and possibly on other continents go unrecognized. The development of an affordable, accessible, and feasible diagnostic test for emergomycosis should be prioritized to enable the diagnosis in places where the disease is widespread and to detect the presence of it elsewhere for epidemiological surveillance.