Cutaneous Invasive Opportunistic Fungal Infections in Dogs Receiving Ciclosporin for Immune‐Mediated Diseases: A Case–Control Study

ABSTRACT

Background

Cutaneous opportunistic invasive fungal infections (OIFIs) are mainly reported in immunosuppressed dogs, especially those receiving ciclosporin (CsA). Studies investigating factors other than CsA that favour cutaneous OIFIs in dogs are lacking.

Hypothesis/Objectives

To identify host factors, clinicopathological data, and therapeutic regimens associated with the development of cutaneous OIFIs in dogs receiving CsA for immune‐mediated diseases.

Animals

Client‐owned dogs treated with CsA, with or without systemic glucocorticoids (GCs) for various immune‐mediated diseases. Dogs were classified as cases if they had developed cutaneous OIFIs during CsA treatment, or as controls if they had not.

Materials and Methods

Cases and controls were matched based on the immune‐mediated disease and the duration of CsA treatment. Signalment, anamnestic and pertinent clinical data as well as selected laboratory parameters, including blood cell count, neutrophil‐to‐lymphocyte ratio (NLR), neutrophil‐to‐monocyte ratio (NMR), C‐reactive protein, haptoglobin, total globulins and immunoglobulin (Ig)G and IgM serum levels, were retrospectively collected and analysed.

Results

Eight OIFI‐affected dogs were matched with 20 control dogs. Among the examined variables, only higher neutrophil count and neutrophil ratios (NLR, NMR) were significantly associated with cutaneous OIFIs.

Conclusions and Clinical Relevance

No significant associations were observed between host factors, treatment regimens, and OIFI development in dogs receiving CsA. Further research is needed to clarify the role of GCs and to evaluate NLR and NMR as monitoring tools in immunosuppressed dogs.

Background: Cutaneous opportunistic invasive fungal infections (OIFIs) are mainly reported in immunosuppressed dogs, especially those receiving ciclosporin (CsA). Studies investigating factors other than CsA that favour cutaneous OIFIs in dogs are lacking. Hypothesis/Objectives: To identify host factors, clinicopathological data, and therapeutic regimens associated with the development of cutaneous OIFIs in dogs receiving CsA for immune‐mediated diseases. Conclusions and Clinical Relevance: No significant associations were observed between host factors, treatment regimens, and OIFI development in dogs receiving CsA. Further research is needed to clarify the role of GCs and to evaluate NLR and NMR as monitoring tools in immunosuppressed dogs.

1. Introduction

Cutaneous opportunistic invasive fungal infections (OIFIs) are caused by common saprophytic fungi that penetrate the skin through traumatic inoculation or wound contamination [1]. In some cases, they develop following the spread of a disease acquired via an alternative route of infection, such as the respiratory or the gastrointestinal tract [1, 2, 3]. Skin lesions are characterised primarily by the presence of single or multiple, intact or ulcerated, cutaneous or subcutaneous nodules, which can be localised or disseminated. They are often found in areas of the body that come into contact with the soil, such as the limbs and the nose [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12].

Despite the lack of epidemiological data, OIFIs are traditionally regarded as rare in immunocompetent dogs [1]. By contrast, the number of reports of OIFIs in dogs treated with immunosuppressive therapies has increased over the last 20 years [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12].

Two retrospective studies in the USA found OIFI prevalence rates of 1.67% and 13%, respectively, in dogs treated with immunosuppressive drugs and systemic glucocorticoids (GCs) for immune‐mediated diseases [11, 12]. Using ciclosporin A (CsA) to treat autoimmune diseases in dogs has been identified as a risk factor for OIFIs [12].

In humans, in addition to the use of T‐cell immunosuppressants and GCs, several risk factors for OIFIs have been identified. These include acquired or innate immunodeficiency, invasive procedures, hospitalisation, haematological malignancies, diabetes, solid organ transplant and autoimmune diseases [13, 14, 15]. Moreover, several host factors, including occupations and hobbies involving outdoor work, such as farming or gardening, have been associated with OIFIs acquired through skin trauma in people [16].

In dogs, factors that favour OIFIs other than immunosuppressants have not been investigated. Cobalamin deficiency and innate immunodeficiency have been suggested as possible underlying conditions of OIFIs in dogs, yet no further studies have been conducted [2, 3]. Identifying other factors associated with OIFIs would help to recognise which dogs treated with CsA are more likely to develop OIFIs.

The aim of this study is therefore to investigate the possible association between selected host factors, clinicopathological data and therapeutic regimens, and the development of OIFIs in dogs receiving CsA for immune‐mediated diseases.

2. Materials and Methods

2.1. Ethics

Clinical and anamnestic information, as well as samples, were collected solely for diagnostic purposes, with the owner's informed consent. All procedures were performed in accordance with good clinical practice and ethical guidance specified in Gazzetta Ufficiale 1996;289:47–53. No personally identifiable data were used.

2.2. Study Design

This case–control study included client‐owned dogs receiving CsA to treat various immune‐mediated diseases, which were evaluated at a referral veterinary hospital in Italy between January 2020 and July 2025. Dogs were classified as cases if they had been diagnosed with cutaneous OIFIs during CsA treatment, or as controls if they had not.

2.3. Selection of Cases

Dogs diagnosed with OIFIs while receiving CsA for an immune‐mediated disease were included in the study as cases if their electronic medical records contained information on medical history, clinical signs, immunosuppressive treatment regimen and blood test results at the time of OIFI diagnosis. Concurrent GCs use was the only additional immunosuppressive therapy allowed.

The diagnosis of cutaneous OIFI required histological demonstration of the fungal organism surrounded by a pyogranulomatous‐to‐granulomatous inflammation [17, 18].

Fungal pathogens were identified at the Parasitology and Mycology Unit of the Istituto Zooprofilattico Sperimentale delle Venezie through culture and PCR, whenever possible (see Methods S1) [19, 20, 21].

2.4. Selection of Controls

Adult dogs that did not develop OIFI while receiving a minimum daily dose of 5 mg/kg of CsA for a duration similar to that of the OIFI cases were evaluated for inclusion as controls (see the Section 2.5). Concurrent GCs were permitted.

Dogs were included as controls if their electronic medical records contained the same clinical and laboratory information collected for the OIFI cases.

2.5. Matching Cases With Controls

OIFI‐affected and control dogs were matched based on the immune‐mediated disease for which CsA was administered and the duration of CsA treatment. If a control case with the same immune‐mediated disease as the OIFI case could not be found, then a case with a comparable disorder was chosen.

For OIFI‐affected dogs that had received CsA for < 3 months, only dogs treated for a similar length of time could be selected as controls. For OIFI cases treated with CsA for > 3 months, a difference of ≤ 2 months duration of CsA therapy between OIFI cases and controls was permitted.

The matching was performed at a ratio of 1:2 or 1:3, depending on the available controls.

2.6. Data Collection

The information retrieved from the electronic medical records for both OIFI cases and controls included signalment, preventive treatments (regular vs. irregular parasite prophylaxis and vaccination), environment (indoors vs. outdoors, presence or absence of cohabiting animals), diet (commercial vs. home‐made), type of immune‐mediated disease for which CsA was administered, and the presence of cutaneous or noncutaneous infections other than OIFIs.

Furthermore, the following data were retrospectively retrieved and recorded for both OIFI cases and controls:

• –dosages of CsA and GCs administered at the beginning of the CsA treatment (T0).
• –dosages and duration of CsA and GCs treatment at the time cases were diagnosed with OIFI (T1).
• –laboratory parameters, selected among those routinely checked in dogs undergoing immunosuppressive therapies and measured at T1. These parameters included haematocrit (Hct), white blood cell (WBC), total and banded neutrophil, lymphocyte, monocyte, eosinophil, and platelet counts (ADVIA 2120i Haematology System; Siemens Healthineers GmbH), C‐reactive protein, haptoglobin, total globulin (Atellica CH930; Siemens Healthineers), and immunoglobulin (Ig)G and IgM serum levels (AU600; Olympus Europe GmbH) [22]. Additionally, neutrophil‐to‐lymphocyte ratio (NLR) and neutrophil‐to‐monocyte ratio (NMR) at T1 were calculated.
• –Neutrophilia [23], increased C‐reactive protein [24], and increased NLR and NMR [25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37] were selected as indicators of inflammation. Anaemia was potentially associated with a chronic inflammatory disease [38]. Neutropenia [36, 39], lymphopenia [40] and reduced Ig concentrations [41, 42] were considered possible indicators of immunosuppression.

2.7. Statistical Methods

Qualitative variables were summarised using counts and percentages and the quantitative ones using means and standard deviations (SDs), or medians and interquartile ranges (IQRs), according to the distributional assumptions. The normality of the distribution of the quantitative variables was assessed using the Shapiro–Wilk test. With normality, Student's t‐test was used to assess significant differences between the groups of subjects, while without normality, the Mann–Whitney rank test was used. Effect sizes were quantified as differences in medians with 95% confidence intervals (95% CI), using the OIFI group as the baseline category. Associations between the qualitative variables were assessed using the exact F‐test. An ANCOVA regression model was applied to evaluate the effects of the covariates OIFI/control groups and GCs therapy dosage on the increase or decrease of the numerical variable NLR. Statistical significance was set at p < 0.05. The data were analysed using R v4.3.2 statistical software.

3. Results

Between January 2020 and July 2025, eight dogs, representing 0.02% (eight of 36,118) of the total hospital canine population, were diagnosed with cutaneous OIFI while receiving CsA to treat an immune‐mediated disease. These dogs were matched with 20 control dogs that were treated with CsA for the same or comparable immune‐mediated diseases. Details on signalment, anamnestic data, and immunosuppressive treatment regimens of OIFI‐affected and control dogs are summarised in Table 1.

TABLE 1.

Demographic, anamnestic and treatment details of the entire study population, the opportunistic invasive fungal infection (OIFI)‐affected dogs and control dogs.

Variables		Overall (n = 28)	OIFI group (n = 8)	Control group (n = 20)	p
Signalment
Breed (%)	Mixed‐breed	6 (21.4)	2 (25)	4 (20)	1.00
	Purebred	22 (78.6)	6 (75)	16 (80)
Sex (%)	M	8 (28.6)	3 (62.5)	5 (25)	0.65
	F	20 (71.4)	5 (37.5)	15 (75)
Sexual status (%)	I	10 (35.7)	3 (37.5)	7 (35)	1.00
	Ne	18 (64.3)	5 (62.5)	13 (65)
Age, months mean (SD)		99 (32)	109 (34)	95 (31)	0.32
Anamnestic data
Environment (%)	Mostly indoor	10 (35.7)	2 (25)	8 (40)	0.67
	Mostly outdoor	18 (64.3)	6 (75)	12 (60)
Cohabiting animals (%)	Yes	14 (50)	6 (75)	8 (40)	0.21
	No	14 (50)	2 (25)	12 (60)
Food (%)	Commercial	20 (71.4)	7 (87.5)	13 (65)	0.37
	Mixed	8 (28.6)	1 (12.5)	7 (35)
Vaccination (%)	Yes	19 (67.9)	4 (50)	15 (75)	0.37
	No	9 (32.1)	4 (50)	5 (25)
Prevention for ectoparasites (%)	Yes	14 (50)	4 (50)	10 (50)	1.00
	No	14 (50)	4 (50)	10 (50)
Prevention for endoparasites (%)	Yes	23 (82.1)	6 (25)	17 (85)	0.61
	No	5 (17.9)	2 (75)	3 (15)
Immunosuppressive treatment, median (IQR)
CsA, mg/kg, T0		10 (9.5–10.4)	10 (9.1–10.6)	10 (9.5–10.4)	1.00
CsA, mg/kg, T1		9.2 (6.8–10.7)	9.3 (6.8–11.2)	9.2 (7.2–10.5)	1.00
CsA duration, months		3 (2–5.6)	3 (1.8–6)	3 (2–5.6)	0.96
GCs, mg/kg, initial a		1.9 (0.9–2)	1.8 (1–2)	1.9 (0.8–2)	0.78
GCs, mg/kg, T0		2 (1–2)	2 (1.5–2.2)	1.6 (0.9–2)	0.37
GCs, mg/kg, T1		0.6 (0.2–1)	1 (0.5–1.2)	0.5 (0–0.9)	0.30
GCs duration, months		3 (1–4.3)	3 (1.8–12)	2.5 (1–4)	0.44

Note: The OIFI‐affected dogs and the matched control dogs were compared using Student's t‐test or the Mann–Whitney rank test, depending on the distribution of the continuous variables. Categorical variables were analysed using Fisher's exact test. A p‐value of < 0.05 was considered statistically significant.

Abbreviations: CsA, ciclosporin; F, female; GCs, glucocorticoids; I, intact; IQR, interquartile range; M, male; Ne, neutered; SD, standard deviation; T0, beginning of the treatment; T1, time of OIFI diagnosis.

^a Before starting CsA.

3.1. Clinical Characteristics of OIFI‐Affected Dogs and Control Cases

In the OIFI‐affected dogs, one of the following immune‐mediated diseases had been diagnosed: inflammatory bowel disease (n = 2), immune‐mediated haemolytic anaemia (n = 2), Evans syndrome (n = 1), myelodysplasia (n = 1), and meningoencephalomyelitis of unknown origin (n = 1). In one case, a diagnosis of presumed immune‐mediated mucocutaneous ulcerative dermatitis was made, yet it was not further investigated.

The median time to develop OIFI after starting CsA was 3 months (IQR 1.8–6).

In the OIFI cases, skin lesions associated with infection consisted of cutaneous nodules (n = 5), nodules and papules (n = 2), and plaques (n = 1). Five cases (83%) presented with ulcerated lesions. One dog had nodules that extended into the subcutaneous tissue. Most dogs (n = 6; 75%) had multiple lesions that in four cases involved different body regions, including neck and thorax (n = 1), trunk and limbs (n = 2), paws and nose (n = 1), trunk, limbs and ear pinnae (n = 1). In one dog, lesions were confined to one foreleg (n = 1) and in another to the dorsal nose (n = 1).

Occasionally, enlargement of the regional lymph nodes (n = 2), fever (n = 1), and lameness (n = 3) were also reported in the medical records.

Upon histopathological examination, fungal hyphae and spores were visible in haematoxylin & eosin samples from seven cases. Their presence was confirmed using the periodic acid Schiff (PAS) reaction and Grocott's methenamine silver (GMS) stain. In one case, the fungal organisms were only detected after the GMS stain was used. In this case, Dydimella exigua was identified.

Fungal identification was obtained in six cases using PCR performed on one or more samples (n = 6) and mycological culture (n = 4). In two cases, fungal species were not identified despite PCR performed on formalin‐fixed paraffin‐embedded (FFPE) tissue (n = 1) or fresh biopsy tissue (n = 1).

After the diagnosis of OIFI, reduction of immunosuppressive therapy was attempted in all cases. Systemic antifungal therapy was initiated in seven dogs. In three cases, this therapy was associated with the surgical removal of lesions. In one case, surgical removal of the fungal lesions was the only therapy performed.

Complete resolution of the lesions was achieved in six cases. In one case, partial resolution of the lesions was observed 3 months after initiation of antifungal therapy; however, the dog was subsequently lost to follow‐up. One dog was euthanised as a consequence of poor control of the primary immune‐mediated disease and progression of OIFI. Details about the OIFI‐affected dogs, including clinical signs, diagnostic procedures, fungal species, treatments and follow‐up, are summarised in Table 2.

TABLE 2.

Signalment, clinical presentation, diagnosis, treatment and outcome of the opportunistic invasive fungal infection (OIFI)‐affected dogs.

Dog	Breed, age (months) and sex	Primary disease	Immunosuppressive therapy (T0)	Immunosuppressive therapy (T1)	Skin lesions	OIFI diagnosis				Antifungal therapy	Medication adjustments	Outcome (months)
						Special stains (HP)	Culture (biopsy collection)	PCR
								Specimen	Result
1	Mixed‐breed, 162, CM	IBD	CsA: 13.2 mg/kg once daily P: 0.9 mg/kg once daily	CsA: 6.3 mg/kg every 72 h P: 0.3 mg/kg once daily	Cutaneous digital nodule	H&E: − PAS: − GMS: +	Not performed	FNA of the nodule	Dydimella exigua	ITR: 10 mg/kg once daily Surgery	CsA: stopped P: ↓50%	CR (10)
2	English cocker spaniel, 88, SF	McUD	CsA: 7 mg/kg once daily	CsA: 7 mg/kg every 48 h	Ulcerated cutaneous nodules on a foreleg	H&E: + PAS: + GMS: +	Not performed	FFPE tissue	Negative	ITR: 10 mg/kg once daily	CsA: stopped	CR (3)
3	French bulldog, 91, SF	ME	CsA: 10 mg/kg once daily P: 2.2 mg/kg once daily	CsA: 11 mg/kg once daily P: 1 mg/kg once daily	Cutaneous and subcutaneous nodules on the neck and thorax	H&E: + PAS: + GMS: +	Not performed	FNA of lymph node	Scedosporium spp.	ITR: 10 mg/kg once daily TER: 30 mg/kg once daily	CsA and P: ↓50%	E (2)
4	Mixed‐breed, 125, CM	IMHA	CsA: 4.4 mg/kg once daily P: 1.7 mg/kg once daily	CsA: 4.4 mg/kg once daily P: 1.7 mg/kg once daily	Ulcerated cutaneous plaques on the nose	H&E: + PAS: + GMS: +	Not performed	Fresh frozen tissue	Negative	Surgery	CsA and P: stopped MM: started	CR (1)
5	American Staffordshire terrier, 77, M	MDS	CsA: 10 mg/kg once daily P: 2 mg/kg once daily	CsA: 10 mg/kg once daily P: 0.5 mg/kg once daily	Ulcerated cutaneous papules and nodules on the pinnae, trunk and limbs	H&E: + PAS: + GMS: +	Paecilomyces. lilacinus	Fresh tissue Fungal colony	Paecilomyces lilacinus	ITR: 5 mg/kg once daily TER: 30 mg/kg once daily	CsA: stopped P: ↓50% and then stopped	CR (7)
6	Zwergpinscher, 102, SF	ES	CsA: 10 mg/kg once daily P: 2.2 mg/kg once daily	CsA: 15.4 mg/kg once daily P: 1 mg/kg once daily	Ulcerated cutaneous nodules/papules on the paws and the nose Oncolysis	H&E: + PAS: + GMS: +	Bipolaris. cynodontis	Fresh tissue Fungal colony	Bipolaris cynodontis	ITR: 5 mg/kg once daily TER: 30 mg/kg once daily	CsA: stopped P: ↑25%	PR (3)
7	Coton de Tulear, 152, SF	IBD	CsA: 12.2 mg/kg once daily Mp: 2.2 mg/kg once daily	CsA: 11 mg/kg once daily Mp: 1 mg/kg once daily	Cutaneous nodule on the nose	H&E: + PAS: + GMS: +	Negative	Fresh tissue	Bipolaris cynodontis	ITR: 5 mg/kg once daily Surgery	CsA: ↓50% q48 Mp: ↔	CR (1)
8	Labrador retriever, 74, F	IMHA	CsA: 9.8 mg/kg once daily P: 2 mg/kg once daily	CsA: 8.6 mg/kg once daily P: 1.8 mg/kg once daily	Ulcerated cutaneous nodules on the trunk and the limbs	H&E: + PAS: + GMS: +	Curvularia spp.	Fresh tissue Fungal colony	Curvularia. geniculata Bipolaris. cynodontis	Surgery ITR: 10 mg/kg once daily	CsA: ↔ P: ↓50%	CR (1)

Abbreviations: −, negative; +, positive; ↓, reduced; ↔, unchanged; CM, castrated male; CR, complete resolution; CsA, ciclosporin; E, euthanasia; ES, Evans syndrome; F, intact female; FFPE, formalin‐fixed paraffin‐embedded; FNA: fine‐needle aspiration; GMS, Grocott's methenamine silver stain; H&E, haematoxylin & eosin stain; HP, histopathological investigation; IBD, inflammatory bowel disease; IMHA, immune‐mediated haemolytic anaemia; ITR, itraconazole; M, intact male; McUD, muco‐cutaneous ulcerative dermatitis; MDS, myelodysplasia; ME, meningoencephalomyelitis; MM, micofenolate mofetil; Mp, methylprednisolone; P, prednisolone; PAS, periodic acid Schiff stain; PR, partial resolution; SF, spayed female; T0, beginning of the treatment; T1, time of OIFI diagnosis; TER, terbinafine.

Seven OIFI‐affected dogs were matched with control dogs treated for the same immune‐mediated diseases. One dog with cutaneous vasculitis and two dogs with sebaceous adenitis were selected as controls for the OIFI case diagnosed with immune‐mediated muco‐cutaneous ulcerative dermatitis. No skin lesions suggesting OIFIs were reported in any of the control dogs.

3.2. Host Factors and Other Infections

No statistically significant differences were found in terms of sex, sexual status, parasite prevention, vaccine prophylaxis, environment and diet between OIFI‐affected dogs and control dogs (Table 1).

Lower urinary tract infections were reported in OIFI cases (37.5%) and control dogs (20%), with no significant associations with either group (Table 1). No other types of infections were reported in either group.

3.3. Immunosuppressive Regimens

Systemic glucocorticoids were administered to seven of eight (88%) OIFI‐affected dogs and 16 of 20 (80%) control dogs in conjunction with CsA. In all cases, GCs had been initiated before T0 and were still administered at T1 in all except two control dogs (Table 1). A significant reduction in the dose of GCs, from immunosuppressive (2 mg/kg) to anti‐inflammatory dosages (≤ 1 mg/kg), was observed in both groups from T0 to T1 (Figure 1).

FIGURE 1.

Comparison between systemic glucocorticoid (GCs) dosages in the opportunistic invasive fungal infection (OIFI)‐affected and control dogs during the observation period. The boxplots show the median daily GCs dose (GCs, thick line) at the beginning of therapy for the underlying immune‐mediated diseases (initial), at the time of the ciclosporin introduction (T0), and at the time of the OIFI diagnosis/matching (T1) for control (C) and opportunistic fungal infection (OIFI) groups. Boxes represent the interquartile range and the whiskers show the range excluding outliers. Significant differences are indicated by *. The level of significance was set at p < 0.05.

No statistically significant differences were observed in the dosage of CsA and GCs or the duration of treatment between the two groups at any time point. However, OIFI‐affected dogs received a higher median dosage of GCs at both T0 and T1 (Figure 1).

3.4. Laboratory Findings

The results of the selected laboratory parameters measured at T1 in OIFI‐affected dogs and in control dogs are shown in Table 3.

TABLE 3.

Laboratory findings of opportunistic invasive fungal infection (OIFI)‐affected and control dogs.

Laboratory parameters	OIFI group (n = 8)	Control group (n = 20)	95% CI for difference	p
(Reference interval, unit)	median (IQR)	median (IQR)
CBC
HCT (39%–59.2%)	41.8 (33.7–45.7)	44 (38.5–49.6)	(−2.8, 13.8)	0.28
WBC (5.45–12.98 × 103/μL)	16.7 (11.4–24.4)	8.2 (7–13.4)	(−14.1, 1)	0.07
Total neutrophils (3555–9314/μL)	13,485 (9660–22,160)	6105 (5078–10,570)	(−13,630, −890)	0.04
Lymphocytes (1169–3810/μL)	1385 (602.5–2552.5)	1650 (1205–1970)	(−1160, 910)	0.65
Monocytes (186–798/μL)	545 (401–910)	545 (370–927.5)	(−370, 330)	0.94
Eosinophils (104–1164/μL)	60 (35–75)	70 (50–140)	(−30, 80)	0.46
PLT (176–479 × 103/μL)	597 (490.8–693)	443 (289.3–521.8)	(−334, 101)	0.12
NLR	7.9 (6.9–11.4)	3.3 (2.7–7.5)	(−7.3, −1.8)	0.02
NMR	19.2 (15.5–28.2)	12.1 (9.9–15.4)	(−16.9, −2.8)	< 0.01
Biochemical profile
CRP (0.01–0.2 mg/dL)	0.3 (0.01–0.7)	0.2 (0.01–0.6)	(−0.5, 0.2)	0.66
Hp (1–96 mg/dL)	362 (234.8–537)	273 (123.3–509.5)	(−254, 106)	0.32
Albumin (2.7–3.6 g/dL)	3 (2.7–3.2)	3.3 (2.9–3.7)	(−0.3, 0.8)	0.24
Total globulins (2.6–3.9 g/dL)	3 (2.8–3.3)	3.2 (3.1–3.4)	(−0.5, 0.5)	0.41
IgG (323–659 mg/dL)	389.5 (349.8–495)	365.5 (303.5–574.3)	(−158, 74)	0.44
IgM (61–99 mg/dL)	90.5 (82.3–101.3)	111.5 (94.8–125.3)	(−1, 35)	0.06

Note: Band and mature neutrophils are reported as total neutrophils. Median values of the laboratory parameters of the OIFI‐affected dogs and the matched control dogs were compared using the Mann–Whitney rank sum test. Differences in medians with 95% confidence intervals (95% CI) were calculated using OIFI group as reference. Reporting 95% CI for the differences in medians allows for an assessment of the precision and clinical reliability of the estimates, indicating how strongly the data support the observed differences. A p‐value of < 0.05 was considered statistically significant.

Abbreviations: CRP, C‐reactive protein; HCT, haematocrit; Hp, haptoglobin; IgG, immunoglobulin G; IgM, immunoglobulin M; NLR, neutrophil‐to‐lymphocyte ratio; NMR, neutrophil‐to‐monocyte ratio; WBC, white blood cell count.

Two of eight (25%) OIFI‐affected dogs showed leucocytosis and neutrophilia with left shift. Neutrophil count (p = 0.04), NLR (p = 0.02) and NMR (p < 0.01) were significantly higher in OIFI‐affected dogs compared to the control dogs. No other significant differences were found.

When considering the correlations between the dose and duration of GCs treatment and WBC, neutrophil and lymphocyte count, as well as NLR or MNR, the correlation between NLR and GCs dosage showed a different trend between the two groups. However, this difference was not statistically significant (Figure 2).

FIGURE 2.

Heatmap of correlation coefficients among leucocytes, neutrophil‐to‐lymphocyte ratio (NLR) and neutrophil‐to‐monocyte ratio (NMR) and dosage and duration of systemic glucocorticoid (GCs) therapy in opportunistic invasive fungal infection (OIFI)‐affected (b) and control dogs (a). Colours represent the magnitude and direction of correlations (blue = positive, red = negative), while the size of the ellipses is proportional to the absolute value of the correlation coefficient. The correlation threshold was set at |rho| > 0.45 for the control group and at |rho| ≥ 0.7 for the OIFI group.

The fitted ANCOVA model showed that both the dosage of GCs and the group status independently increased the NLR, with a significant interaction (Figure 3). This suggests that variation in the dosage of GCs alone cannot fully account for differences in NLR.

FIGURE 3.

Scatter plot illustrating the association between neutrophil‐to‐lymphocyte ratio (NLR) and systemic glucocorticoid (GCs) dose, with the estimated regression model in the control and opportunistic invasive fungal infection (OIFI) group. Fitted ANCOVA regression model: NLR = 1.17 + 7.69*dose in control group and NLR = 16.19–5.06*dose in OIFI group. Both the marginal effect of the daily GCs dose (p < 0.001) and the effect of group status (p < 0.001) were significant. Additionally, the interaction between OIFI group and dose was significant (p = 0.0018), indicating a potential modification by group of the effect of dose on NLR. The coefficient of determination was R ² = 0.53 (p = 0.0003).

4. Discussion

This study examined the potential association between specific host factors, immunosuppressive regimens, and the development of cutaneous OIFIs in dogs treated with CsA for immune‐mediated diseases.

None of the examined variables were significantly associated with OIFIs, except for the neutrophil count and NLR. These results suggest that among dogs treated with CsA, those that develop OIFIs have no additional predisposing factors and that OIFIs occur by chance. However, the absence of significant associations in this study may be a consequence of the small sample size and the lack of comprehensive bloodwork to compare before any immunosuppressive therapy. Additionally, owing to the retrospective nature of the study, information about behavioural habits or specific outdoor activities potentially associated with a higher risk of OIFIs was unavailable [16]. Future studies involving larger cohorts of dogs should include more detailed investigations of their living environments and behavioural habits.

Because no significant differences were observed in the immunosuppressive treatments that they received, we assume—on the one hand—that the OIFI‐affected dogs and the control dogs experienced the same level of immunosuppression. This also is supported by the lack of significant differences in median lymphocyte count, serum IgG and IgM levels, and the absence of other infections, other than lower urinary tract infections. On the other hand, the immunosuppressive activity of CsA is difficult to predict owing to variability in its pharmacokinetics and pharmacodynamics, which leads to significant differences in drug exposure between dogs [43, 44]. Therefore, even though they received similar doses of the drug, dogs with OIFI may still have experienced a higher degree of CsA‐induced immunosuppression than control dogs. A better way to determine the extent and duration of CsA‐induced T‐cell suppression would have been to monitor the expression of T‐cell cytokines after oral dosing [44]. Unfortunately, this was not feasible owing to the retrospective clinical nature of the study.

Most published cases of OIFIs in immunosuppressed dogs report the use of CsA in conjunction with GCs [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12]. In humans, treatment with GCs is a risk factor for invasive fungal infections [15, 16, 17, 18, 19, 20, 21, 22, 45, 46, 47, 48]. In the present study, an association between the administered dose of GCs and the development of OIFIs was not found, even though a trend towards higher doses of GCs was observed in OIFI‐affected dogs compared to control dogs. It is important to note that the small sample size may have prevented the identification of an association between the administered dose of GCs and the development of OIFIs.

Haematological ratios have been studied in humans as potential diagnostic and prognostic markers for infectious, immune‐mediated, and neoplastic diseases [49, 50, 51]. Likewise, neutrophil ratios have been evaluated in dogs with inflammatory, infectious, and neoplastic diseases [25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35]. An increase of NLR has been suggested as an inflammatory marker and as a supportive biomarker and potential monitoring tool for hyperadrenocorticism in dogs [25, 32]. The usefulness of NLR in dogs with skin diseases has not yet been investigated. In the present study, the NLR of OIFI‐affected dogs was higher than that reported for healthy dogs [29, 33, 37]. Furthermore, higher values were observed in dogs with OIFI than in control cases. This may be attributed to several factors, including an inflammatory response to the fungal infection, a more severe underlying immune‐mediated disease, or greater impact of GCs on neutrophil and lymphocyte counts in OIFI dogs compared to control dogs [25, 33]. Interestingly, when we evaluated the influence of GCs treatment on leucocyte ratios, OIFI cases showed an opposite trend compared to control cases. As the GCs dose decreased, a reduction in the NLR was observed in control cases owing to the expected decline in neutrophil counts and increase in lymphocyte counts. Conversely, in OIFI cases, an increase in the NLR was seen as the GCs dosage was decreased. This suggests that the variation in GCs dosage is not the only factor affecting the NLR in the OIFI group. Whether OIFI itself or a poorly controlled underlying immune‐mediated disease significantly affects the NLR in OIFI cases remains to be established. Further studies are warranted to investigate the utility of neutrophil ratios as markers for monitoring and tailoring immunosuppressive treatment and its complications in dogs.

This study also provides new valuable insights into canine cutaneous OIFIs. First, it supports the notion that cutaneous OIFIs are rare in the general hospital population (0.02% of the total canine hospital population in this study) [1]. Second, it describes a previously unreported case of cutaneous infection caused by D. exigua . The family Didymellaceae includes coelomycetous fungi, which are widely distributed and mainly related to plants; however, they are rarely reported as human pathogens [45, 46].

Finally, this study adds clinically relevant information to the limited literature on OIFIs in dogs. In particular, it highlights the importance of regular and careful examinations of the entire skin surface, especially the extremities, in dogs undergoing CsA treatment, as OIFIs can develop at any time during therapy.

The findings of this study should be regarded as preliminary because of several limitations.

The small sample size in this case‐control study limits its statistical power, reducing the ability to detect meaningful and robust associations. For the same reason, the significant estimates obtained are likely to be affected by a degree of imprecision, as indicated by the wide 95% CI.

5. Conclusions

In conclusion, this study found no significant associations between the selected host factors, therapeutic regimens, and the development of OIFIs in dogs treated with CsA for immune‐mediated diseases. Further investigations are warranted to assess the potential role of GCs as an additional risk factor for OIFIs in dogs receiving CsA. Moreover, the potential utility of NLR as a monitoring tool in immunosuppressed dogs should be further explored.

Author Contributions

Laura Ventura: formal analysis, writing – review and editing, writing – original draft. Patrizia Danesi: investigation, writing – review and editing. Michela De Lucia: conceptualization, investigation, writing – original draft, writing – review and editing, methodology, supervision, data curation, validation. Carolina Mendes: conceptualization, data curation, writing – review and editing, investigation. Erika Carli: conceptualization, formal analysis, writing – original draft, writing – review and editing, investigation. Laura Berto: data curation, conceptualization, investigation, writing – review and editing. Paola Orlandini: data curation, writing – original draft, conceptualization, investigation, methodology, validation, writing – review and editing.

Funding

San Marco Veterinary Clinic and Laboratory, Veggiano, Padua, Italy.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Methods S1: Mycological culture and molecular Identification from skin biopsy samples.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.