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.