DISCUSSION
This report highlights a case of a dog on multimodal immunomodulating therapy for IMHA that became coinfected with Aspergillus citrinoterreus and Curvularia lunata . Our case emphasizes the importance of vigilant monitoring for adverse effects related to immunosuppression and opportunistic infections, and it demonstrates the necessity of appropriate and adequate evaluation of lesions that arise after initiation of immunosuppression treatment protocols.
Opportunistic fungal infections in small animals were recently described in a review article by Dedeaux, et al. In that review, they describe how opportunistic mycoses caused by soil saprobes that are typically non-pathogenic to immunocompetent animals have emerged as clinically relevant causes of disease in patients undergoing immunomodulating therapy for immune-mediated diseases. The same review also mentions naturally occurring causes of immunocompromise that can predispose patients to opportunistic mycoses, with examples includingCandida spp. urinary tract infection in diabetic dogs and cats, disseminated aspergillosis and hyalohyphomycosis in German shepherd dogs with suspected familial immunodeficiency, Pneumocystis pneumonia in miniature dachshunds with combined variable immunodeficiency, systemic mold infection in young dogs with hereditary cobalamin deficiency, and phaeohyphomycosis in cats with diabetes mellitus, retrovirus infection, or lymphoid neoplasia.9Opportunistic mycoses in animals with naturally occurring causes of immunocompromise are rare and generally well-described. Opportunistic mycoses in patients receiving immunomodulating therapies are more likely to be caused by saprobic organisms that can be more difficult to definitively differentiate.9 One report describes an incidence of cutaneous opportunistic fungal infection in dogs receiving immunosuppressive therapy of up to 13%.10
Aspergillus species are saprobic organisms ubiquitously found in the environment and are opportunistic pathogens. Aspergillus fumigatus , A. flavus , A. terreus , A. niger , andA. deflectus cause disease in dogs. A. fumigatus andA. flavus usually cause localized disease in the nasal cavity while A.terreus is more commonly associated with systemic aspergillosis in dogs. However, A. deflectus and A. nigerhave been reported to cause disease in some dogs.11
Aspergillus citrinoterreus was first described by Guinea et al as a new species of Aspergillus section Terrei in 2015.5It is closely related to A. terreus and differs in its colony characteristics and microscopic structure. A clinically significant note is Aspergillus citrinoterreus was found to be more susceptible to azole antifungals compared to A. terreus , but both species still showed clinical resistance to amphotericin B.5 Little is known about the geographical distribution of Aspergilluscitrinoterreus . Species in the Aspergillus sectionTerrei are reported as common pathogens of invasive aspergillosis in humans.12-14 Infection with Aspergillus citrinoterreus has not been reported in the dog.
Phaeohyphomycosis is an infrequent infection in both humans and animals, though it has been more frequently reported in recent years in immunosuppressed patients (most commonly in solid organ transplant patients).9,15 It is the name given to cutaneous and systemic diseases caused by black molds that develop dark-walled, septate mycelia in tissue.16 These infections are usually cutaneous and acquired traumatically, typically affecting exposed areas of the head or upper limbs, though disseminated cases are reported. Phaeohyphomycosis is an overall term used to describe infection from one of any of over 60 genera of dematiaceous (pigmented) fungi. Agents identified to cause infection in veterinary patients include Alternaria, Aureobasidium, Bipolaris/Curvularia, Cladophialophora, Exophiala, Fonsecaea, Lecythophora, Microsphaeropsis, Moniliella, Mycoleptodiscus (Muyocopron ), Phialophora, Ramichloridium, Scolecobasidium , Scytalidium , andUlocladium (Alternaria ).9,17-19 These fungi are saprobic, widely-distributed organisms found in soil, water, and decaying vegetable matter, which are usually non-pathogenic, but can be devastating to immunocompromised hosts.
The most common clinical manifestations of phaeohyphomycosis in small animals are lesions associated with the digits, pinnae, nasal planum, or the nasal cavity in cats. However, multifocal cutaneous lesions more commonly occur in immunocompromised patients or dogs treated with multiple immunosuppressive agents. Furthermore, the multifocal presentation is even more common when cyclosporine is one of the immunosuppressive agents used.17 One report describes an odds ratio of 7.1 for patients diagnosed with cutaneous opportunistic fungal infection that were being treated with cyclosporine.10
Phaeohyphomycosis in humans is divided into four forms based on the location of the infection and route of inoculation. These include superficial, cutaneous, subcutaneous, and systemic.16The most common types of phaeohyphomycosis in veterinary medicine are the subcutaneous and systemic forms.20 In most of the cases reported in domestic animals, there is no involvement of the epidermis or upper dermis, and traumatic implantation or wound contamination is thought to be the primary mode of infection.16
Diagnosis of fungal infections requires microscopic detection of intralesional fungal elements, culture, and often additional diagnostic, such as serology or molecular diagnostics.21 The utility of cytology is dependent on sampling and processing techniques, exfoliation of representative populations and extracellular elements, and degree of heterogeneity present within the sample, which can limit its accuracy. However, cytology can provide rapid, preliminary information that can be helpful in determination of subsequent diagnostic follow-up; this is especially true for infectious lesions. It should be noted that microscopic evaluation, whether cytologic or histopathologic, is not recommended as a stand-alone diagnostic tool to determine fungal identification. Culture, isolation, and molecular characterization (PCR, genome sequencing) are required for speciation.21 Morphological diagnosis of fungal species is additionally difficult for reasons such as the presence of cryptic species within different genera, the absence of morphological structures that aid in identification, the lack of expertise in fungal identification in clinical microbiology laboratories and the failure for the fungus to grow from clinical specimens. Molecular testing methods using polymerase chain reaction and comparative sequence analysis enable definitive identification of most clinical isolates.22For aspergillosis, diagnosis of fungal infection is achieved through identification of fungal hyphae within tissue samples or urine or by detection of the fungal cell wall antigen galactomannan in blood or urine as well as DNA sequencing of the isolated fungus from specimens.23
Diagnosis of phaeohyphomycosis requires evidence of pigmented fungi in wet mounts or in histologic sections in addition. However, due to similar histologic appearance of many phaeohyphomycotic agents, definitive diagnosis is made by culture and specific identification of the etiologic agent.16 Local disease may be cured with excision of diseased tissue, but systemic disease can often be deadly as the disease can often be refractory to therapy.5
A comprehensive review of literature reporting disseminated phaeohyphomycosis infections in people identified a total of 72 cases from 1966 to 2002.24 In that review, the prognosis is noted to be particularly poor with an overall mortality rate of 79%. Within the cases reported in that review, several treatments were attempted, but none were associated with improved survival. There is one report in veterinary medicine of successful treatment of disseminated cutaneous phaeohyphomycosis in a dog caused by Curvularia lunata . That case was treated with a combination of amphotericin B and itraconazole. Interestingly, infection in that case was also suspected to be secondary to immunosuppression with a combination of glucocorticoids and cyclosporine.25
The patient in the current report was being treated with immunosuppressive doses of both cyclosporine and prednisone for the management of IMHA. It is likely this therapy caused sufficient immune suppression to induce susceptibility to opportunistic fungal infections. The clinical history provided, in combination with laboratory data, and the gross and microscopic disease, suggest the route of infection was likely through the cutaneous wound followed by hematogenous spread to the previously listed organs. Identification of Aspergillus in the pulmonary tissue and microscopic findings of pneumoconiosis and eosinophilic edema are suggestive of inhalation and potential for concurrent direct inhalational disease, and further visceral spread cannot be completely excluded.
This report highlights a case of coinfection in a dog with two fungal organisms–Aspergillus citrinoterreus and Curvularia lunata– likely due to multimodal immunomodulating therapy for presumptive non-associative IMHA. Additionally, this is the first reported case of disseminated Aspergillus citrinoterreusinfection in a dog in North America to the authors’ knowledge. Cases ofAspergillus terreus species infections are important to note as they may carry a poorer prognosis due to clinical resistance to Amphotericin B and higher mortality rates.26 This patient developed a severe disseminated infection of Aspergillus citrinoterreus in addition to a localized Curvularia infection that both led to considerable morbidity and eventually euthanasia. As the two fungal species were collected from different body sites on this patient, this case also highlights the importance of thorough evaluation for possible coinfections in these immunocompromised patients.