Temporal tracheobronchial lymphadenopathy resolution is affected by initial severity and poorly correlated to serologic antibody testing in dogs with pulmonary coccidioidomycosis

Rebecca Urion Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH

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Jared A. Jaffey Department of Specialty Medicine, College of Veterinary Medicine, Midwestern University, Glendale, AZ

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Eric T. Hostnik Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH

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Andrew S. Hanzlicek MiraVista Diagnostics, Indianapolis, IN

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George E. Moore Department of Veterinary Administration, College of Veterinary Medicine, Purdue University, West Lafayette, IN

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Abstract

OBJECTIVE

To determine associations between antibody serologic tests and tracheobronchial lymphadenopathy (TBL) in dogs with pulmonary coccidioidomycosis and identify variables associated with time to resolution of TBL.

ANIMALS

32 client owned dogs with newly diagnosed pulmonary coccidioidomycosis from October 2020 to February 2021.

METHODS

Prospective cohort study. Thoracic radiographs and anti–Coccidioides spp antibody serology were performed at baseline and once every 3 months until remission or for a maximum of 12 months. Radiographic tracheobronchial lymph node height, length, and area were measured and recorded as ratios via comparison with the length of the T4 vertebral body (LT4) and length of the manubrium. Severity of TBL was also subjectively categorized as mild, moderate, or severe.

RESULTS

Tracheobronchial lymphadenopathy was identified in 81% (26/32; 95% CI, 64% to 93%) of dogs. There was no relevant association between TBL presence or severity and antibody serology results. Tracheobronchial lymphadenopathy resolved in 72% (n = 18) of dogs at the 3-month evaluation. The median time to resolution of TBL after initiation of fluconazole was 96 days (range, 72 to 386 days). Univariate analysis identified increasing TBL severity (hazard ratio, 0.40; 95% CI, 0.19 to 0.84; P = .02) and length:LT4 ratio (hazard ratio, 0.41; 95% CI, 0.20 to 0.82; P = .01) as variables associated with reduced probability of resolution of TBL.

CLINICAL RELEVANCE

Antibody serologic test results are not clinically useful to predict TBL presence or severity in dogs with pulmonary coccidioidomycosis, and larger tracheobronchial lymph nodes are more likely to take longer to resolve. Resolution of TBL occurs in most dogs within 3 to 6 months after fluconazole administration.

Abstract

OBJECTIVE

To determine associations between antibody serologic tests and tracheobronchial lymphadenopathy (TBL) in dogs with pulmonary coccidioidomycosis and identify variables associated with time to resolution of TBL.

ANIMALS

32 client owned dogs with newly diagnosed pulmonary coccidioidomycosis from October 2020 to February 2021.

METHODS

Prospective cohort study. Thoracic radiographs and anti–Coccidioides spp antibody serology were performed at baseline and once every 3 months until remission or for a maximum of 12 months. Radiographic tracheobronchial lymph node height, length, and area were measured and recorded as ratios via comparison with the length of the T4 vertebral body (LT4) and length of the manubrium. Severity of TBL was also subjectively categorized as mild, moderate, or severe.

RESULTS

Tracheobronchial lymphadenopathy was identified in 81% (26/32; 95% CI, 64% to 93%) of dogs. There was no relevant association between TBL presence or severity and antibody serology results. Tracheobronchial lymphadenopathy resolved in 72% (n = 18) of dogs at the 3-month evaluation. The median time to resolution of TBL after initiation of fluconazole was 96 days (range, 72 to 386 days). Univariate analysis identified increasing TBL severity (hazard ratio, 0.40; 95% CI, 0.19 to 0.84; P = .02) and length:LT4 ratio (hazard ratio, 0.41; 95% CI, 0.20 to 0.82; P = .01) as variables associated with reduced probability of resolution of TBL.

CLINICAL RELEVANCE

Antibody serologic test results are not clinically useful to predict TBL presence or severity in dogs with pulmonary coccidioidomycosis, and larger tracheobronchial lymph nodes are more likely to take longer to resolve. Resolution of TBL occurs in most dogs within 3 to 6 months after fluconazole administration.

Introduction

Coccidioidomycosis, also known as Valley Fever, is a fungal disease primarily found within the southwestern US, with up to 80% of affected dogs in endemic areas having primary pulmonary infection.1,2 Aside from clinical signs related to the respiratory tract disease, dogs with pulmonary coccidioidomycosis might demonstrate nonspecific signs such as decreased appetite, weight loss, fever, and lethargy.3,4

The diagnosis of pulmonary coccidioidomycosis in dogs is typically based upon a collective interpretation of clinical signs, thoracic imaging findings, and anti–Coccidioides spp antibody test results. This diagnostic approach is imperfect and can result in both missed and erred diagnoses. Tracheobronchial lymphadenopathy (TBL) is a common radiographic abnormality in dogs with pulmonary coccidioidomycosis and is reported to occur in 23% to 61% of dogs.4,5 A previous retrospective study identified an association between the presence of radiographic hilar lymphadenopathy and pulmonary coccidioidomycosis in dogs from endemic regions.6 However, radiographic TBL can also be identified in other pulmonary diseases.716

Antibody serology is useful in determining whether a dog has been infected but cannot definitively support that Coccidioides spp are the cause for clinical signs, as clinically and subclinically affected dogs may have overlap in serum titer values.17 Moreover, some dogs with coccidioidomycosis can have low or negative antibody serology results because of a mild infection, delayed seroconversion, or immunodeficiencies.3,4 Establishment of a relationship between antibody serologic testing and TBL presence and severity might be useful to support the relative likelihood and severity of pulmonary coccidioidomycosis.

There is a lack of information regarding the expected temporal response of TBL after initiation of antifungal treatment in dogs. If an expected timeline for TBL resolution in dogs receiving antifungal treatment can be established, this might serve as an additional means of determining whether the initial antifungal choice is appropriate or whether switching to a different medication should be considered in cases with protracted or worsening lymphadenopathy. Furthermore, if associations between baseline variables and time to resolution of TBL are identified, our overall understanding of factors influencing expected duration of antifungal treatment would be improved.

The objectives of this study were to (1) determine whether associations exist between anti–Coccidioides spp antibody serologic test results and radiographic TBL presence or severity in dogs with a clinical diagnosis of pulmonary coccidioidomycosis and (2) determine whether any baseline variables are associated with time to resolution of radiographic TBL after initiation of fluconazole treatment. We hypothesized that 1 or more anti–Coccidioides spp antibody test results would be associated with the presence or severity of radiographic TBL in dogs with pulmonary coccidioidomycosis. In addition, we hypothesized that 1 or more baseline variables would be associated with time to resolution of radiographic TBL.

Methods

Criteria for selection of cases

Client-owned dogs that had a new diagnosis of pulmonary coccidioidomycosis between October 2020 and February 2021 were eligible for inclusion in this prospective study. Dogs were included in the study after informed owner consent was obtained. This study was conducted in accordance with guidelines for clinical studies and approved by the Midwestern University Animal Care and Use Committee (protocol No. 3024). Diagnostic testing for coccidioidomycosis and antifungal treatment decisions were made by the attending clinician, not the research investigators. Serological testing for immunoglobulin (Ig) M and IgG against Coccidioides spp was performed by agar gel immunodiffusion (AGID) and enzyme immunoassay (EIA) through a commercial laboratory (MiraVista Diagnostics). Dogs must have demonstrated 1 or more clinical signs associated with respiratory tract disease (eg, cough, wheeze, increased respiratory effort, exercise intolerance, tachypnea, cyanosis, or syncope), had 1 or more positive serum anti-Coccidioides antibody test result (eg, AGID IgM, AGID IgG, or EIA IgG), and had a minimum of 2-view thoracic radiographs available for review. Dogs with unremarkable thoracic radiographs were still eligible for inclusion if there was a strong clinical suspicion for pulmonary coccidioidomycosis in conjunction with adequate improvement in clinical signs and antibody serologic test results after initiation of antifungal treatment. A positive EIA IgG was defined as results that were ≥ 10 EIA units (EU). Positive AGID IgM or IgG was defined as detectable antibodies in an undiluted serum sample. If positive, serial dilutions were tested (up to 1:128), and the highest dilution with detectable IgG antibodies was reported as the final serum titer result. The upper quantifiable range of the EIA IgG was 80 EU. Exclusion criteria included clinical findings suggestive of concurrent disseminated disease (ie, outside the pulmonary system), the presence of any comorbidities that may cause immune dysregulation (eg, diabetes mellitus, hyperadrenocorticism, neoplasia), administration of any immunosuppressive medications or chemotherapy, or if there had been a historical diagnosis of coccidioidomycosis. Dogs were also excluded if they had been administered antifungal treatment for > 7 days before enrollment. Dogs with unremarkable thoracic radiographs at baseline were subsequently excluded if there was inadequate improvement in clinical signs and antibody serologic test results after initiation of antifungal treatment.

Study design

The study was performed as a prospective cohort study. Dogs were presented for evaluation once every 3 months after initiation of an FDA-approved generic formulation of fluconazole until remission was achieved or for a maximum of 12 months. Serial evaluations were performed in all dogs, regardless of whether radiographic TBL was present at baseline. Three-view thoracic radiographs and antibody serology were performed at each evaluation. Clinical status (ie, subclinical or clinical) was determined at each visit based on the presence or absence of signs related to respiratory tract disease. The attending clinician, not the research investigators, made all other diagnostic and treatment decisions. Remission was defined as the first visit at which the following criteria were met: (1) anti-Coccidioides IgG titer of ≤ 1:8, (2) thoracic radiographs that were either unremarkable or static mild disease without TBL compared with previous evaluation, and (3) dogs were subclinical. Medical records were reviewed for each dog enrolled. The age, sex, weight, and breed were recorded. The following clinical information associated with the baseline evaluation was extracted when available: clinical signs (type and duration), medications, respiratory rate, and temperature.

Thoracic radiography

Thoracic radiographs were independently evaluated with commercial software (Horos; Nimble Co LLC) for the presence or absence of increased perihilar soft tissue by 2 board-certified veterinary radiologists (RU, ETH). Radiologists were not blinded to the suspected diagnosis of pulmonary coccidioidomycosis at the baseline evaluation, and as such, the radiographic studies were reviewed as a diagnostic team and a full consensus was reached for each case to minimize the effects of subjective evaluation and best mimic a realistic clinical scenario. However, radiologists were blinded to clinical information and serologic antibody test results at all subsequent evaluations. The presence of carina and/or principal bronchial displacement suggestive of a mass effect was used as an indirect indicator supportive of the presence of TBL. As radiographic interpretation of the perihilar region is inherently complicated due to the complexity of the regional anatomy and radiographic quality factors, the severity of TBL was evaluated using both a subjective and semiobjective classification scheme to maximize overall accuracy of the interpretation. TBL was subjectively characterized as mild, moderate, or severe by means of the following criteria: (1) mild: perihilar soft tissue opacity localized immediately around the carina without deviation of the airways; (2) moderate: perihilar soft tissue that extends away from the tracheal carina and causes slight deviation of the carina and principal bronchi; and (3) severe: a large amount of perihilar soft tissue extending away from the tracheal carina causing a large amount of tracheal carina and principal bronchial deviation (Figure 1). Complete resolution of TBL was considered when there was no longer abnormal soft tissue opacity in the perihilar region of the tracheobronchial lymph nodes. Partial resolution was considered if perihilar soft tissue persisted but was reduced in size when compared with the most recent previous examination.

Figure 1
Figure 1

Representative right lateral thoracic radiographs of client-owned dogs with newly diagnosed pulmonary coccidioidomycosis in a prospective cohort study conducted between October 2020 and February 2021. The images depict the subjective grading of tracheobronchial lymphadenopathy (TBL) severity, which was categorized as (A) mild, (B) moderate, and (C) severe. Dashed line represents outline of tracheobronchial lymph nodes. Note ventral displacement of the carina and principal bronchi in cases graded moderate or severe.

Citation: Journal of the American Veterinary Medical Association 261, 11; 10.2460/javma.23.04.0220

TBL was also semiobjectively measured on lateral radiographs for height and length (Figure 2). The lateral projection (right vs left) on which the perihilar soft tissue was most easily seen and/or well-defined was used for measurement purposes. From these measurements, tracheobronchial lymph node area was calculated using the formula
article image
For tracheobronchial height, length, and area, ratios were performed via comparison with the length of the T4 vertebral body (LT4) and length of the manubrium (LM). These landmarks were selected because they are easily recognizable midline anatomic structures. Both the T4 vertebral body and manubrium were used as comparisons in all cases with TBL in an effort to account for osseous anatomical differences between various breeds and to minimize effects of forelimb superimposition or excessive collimation. Tracheobronchial measurements and the subsequently developed ratios were made to allow for internal control for future evaluations as well as to account for differences in radiographic magnification, patient size, and conformation.18,19
Figure 2
Figure 2

Representative left lateral thoracic radiograph from a dog with newly diagnosed pulmonary coccidioidomycosis. The image illustrates the technique for semiobjective grading of TBL, manubrium, with the yellow and orange lines representing TBL length and height, respectively. The green and purple lines demonstrate measurement of the length of the fourth thoracic vertebra (T4) and manubrium, respectively. The TBL area was compared with both the length of T4 and the manubrium to generate ratios intended to account for differences in dog size and body conformation.

Citation: Journal of the American Veterinary Medical Association 261, 11; 10.2460/javma.23.04.0220

Statistical analysis

Statistical analyses were conducted using commercial STATA SE, version 17.0 (StataCorp) and SigmaPlot (Systat Software Inc). Anti–Coccidioides spp IgG antibody titers quantified by AGID were converted to inverse titers and considered nonparametrically distributed numerical data. When the measured AGID IgG titer and EIA IgG concentration were above the upper limit of detection for the respective assay, data were recorded as the upper limit of detection for statistical purposes. Correlation between inverse IgG titers and other numerical data was assessed by Spearman’s rank correlation coefficient (rs). Correlation between binary test results and other numerical data was assessed by point-biserial correlation (rPB). Associations between variables and TBL as an ordinal outcome (absent, mild, moderate, or severe) were assessed via an ordered logit model. Radiographic resolution of TBL and its association with other variables was assessed by time-to-event analysis with Kaplan-Meier curves and Cox proportional hazard regression models. An α error rate of 5% was designated, and P values of < .05 were considered statistically significant.

Results

Dogs

Thirty-two dogs were included in this prospective cohort study. Two dogs were switched from FDA-approved generic formulations of fluconazole to other antifungal medications. One dog was switched to compounded fluconazole at the 3-month evaluation (day 90) at the owner’s request and was subsequently lost to follow-up. The remaining dog was transitioned to amphotericin B lipid complex (Abelcet) and itraconazole (Sporonox) at the 3-month evaluation (day 93) because of refractory disease. Data from these 2 dogs were retained for all analyses but final time to resolution of TBL. The mean weight was 22.8 kg (SD, 13.1 kg) and median age was 4.4 years (IQR, 6.4 years). There were 19 purebred dogs and 13 mixed-breed dogs. Purebred dogs included Chihuahua (n = 3), Rhodesian Ridgeback (3), American Pit Bull Terrier-type dogs (2), and 1 each of Australian Cattle Dog, American Foxhound, Belgian Malinois, Border Collie, English Bulldog, English Mastiff, German Shorthaired Pointer, Golden Retriever, Labrador Retriever, Miniature Poodle, and Queensland Heeler. The sex distribution was as follows: castrated male (n = 12 [38%]), spayed female (12 [38%]), intact female (5 [16%]), and intact male (3 [9%]).

Clinical information

The distribution of clinical signs at presentation was as follows: cough (n = 31 [97%]), decreased appetite (15 [47%]), lethargy (14 [44%]), exercise intolerance (5 [16%]), tachypnea (5 [16%]), diarrhea (4 [13%]), wheeze (3 [9%]), increased respiratory effort (2 [6%]), sneeze (2 [6%]), vomiting (2 [6%]), and weight loss (1 [3%]). The median duration of time that clinical signs were apparent before presentation was 14 days (IQR, 29.5 days). Accurate respiratory rate data were not obtained in 7 dogs because of panting. The mean respiratory rate on initial presentation was 35.6 breaths/min (SD, 8.8 breaths/min). Temperature data were not available in 3 dogs because of compliance difficulties. Rectal temperatures were ≥ 39.4 °C in 66% (19/29) of dogs on presentation. The mean rectal temperature was 39.6 °C (SD, 0.6 °C). Overall, the mean follow-up time for dogs (n = 31) in the study period was 283.2 days (SD, 114.8 days)

All dogs were initially treated with an FDA-approved generic formulation of fluconazole. The median per os fluconazole dose prescribed was 16.2 mg/kg/d (IQR, 5.3 mg/kg/d). Eight dogs received prednisone after diagnosis, with a mean per os dosage of 0.7 mg/kg/d (SD, 0.27 mg/kg/d). Of these, the mean duration of treatment with prednisone was 11.9 days (SD, 3.4 days).

Baseline antibody serology results

Thirty-one percent (10/32) of dogs had positive AGID IgM results. Ninety-four percent (30/32) of dogs had positive AGID IgG titer results. Semi-quantitative IgG titers ranged from negative to ≥ 1:128. One dog had an AGID IgG titer of ≥ 1:128. The median AGID IgG titer in dogs with positive results was 1:16 (n = 30). Sixty-five percent (21/32) of dogs had positive EIA IgG results, with 2 dogs having results > 80 EU. The mean EIA IgG for 21 dogs with positive results was 41.1 EU (SD, 23.3). One of the dogs with negative AGID IgG had positive IgM and an EIA IgG of 63.3 EU. The other dog with negative AGID IgG had a negative AGID IgM and an EIA IgG of 25.1 EU.

Baseline thoracic radiographs

On baseline radiographs, 44% (14/32) of dogs had evidence of pulmonary disease. Seventy-nine percent (11/14) of these dogs had a solitary pulmonary pattern, of which a bronchial pattern was most common (64% [7/11]), followed by unstructured interstitial pattern (18% [2/11]) and 1 each of structured interstitial pattern and alveolar pattern. Twenty-one percent (3/14) of dogs with pulmonary parenchymal abnormalities had concurrent bronchial and unstructured interstitial patterns. One patient had mild pleural effusion, and no dogs had sternal or cranial mediastinal lymphadenopathy. TBL was present in 78% (14/18) of the dogs with unremarkable pulmonary parenchyma. The remaining 4 dogs had unremarkable thoracic radiographs. Baseline AGID IgG titers in the 4 dogs with unremarkable thoracic radiographs ranged from 1:4 to 1:64. Each dog was treated with fluconazole and had resolution (n = 3) or near resolution (1) of clinical signs related to respiratory tract disease. In addition, AGID IgG titers became negative or decreased by ≥ 2-fold dilution compared with baseline.

Baseline TBL

TBL was present in 81% (26/32; 95% CI, 64% to 93%) of dogs on baseline thoracic radiographs and, when present, was mild (50% [13/26]), moderate (38% [10/26]), or severe (12% [3/26]). The semiobjective TBL measurements were as follows: height:LM (median, 0.63; IQR, 0.47; n = 23), length:LM (median, 1.12; IQR, 0.53; 23), area:LM (median, 19.74; IQR, 23; 23), height:LT4 (median, 1.16; IQR, 0.85; 24), length:LT4 (median, 2.22; IQR, 1.23; 24), and area:LT4 (median, 41.27; IQR, 65.56; 24). Tracheobronchial lymph node measurements were not available for some dogs because the anatomical structures required to determine ratios were not clearly visualized.

Longitudinal assessment of TBL

There were 25 dogs with TBL present at baseline that were treated with an FDA-approved generic formulation of fluconazole throughout the course of the study. One of the 26 dogs that had TBL at baseline was not included in longitudinal assessments because of a transition to an alternate antifungal regimen. TBL resolved in 72% (18/25; 95% CI, 51% to 88%) of dogs at the 3-month evaluation (median, 94 days; IQR, 8). TBL resolved in an additional 2 dogs (8%) by the 6-month evaluation (median, 177 days; IQR, 6), in 3 dogs (12%) by the 9-month evaluation (median, 298 days; IQR, 40), and in 1 dog (4%) by the 12-month recheck (median, 386 days). TBL substantially improved but failed to resolve completely in 1 dog (4%) within the study period. The severity of TBL did not worsen over its baseline status in any affected dogs. Overall, the median time to resolution of TBL was 96 days (range, 72 to 386 days; Figure 3). Time to resolution of TBL based on subjective severity classification was summarized (Supplementary Table S1). There were 6 dogs that did not have radiographic TBL at baseline. One of these dogs was switched to compounded fluconazole and was lost to follow-up. Radiographic TBL did not develop in any of the remaining 5 dogs within a median follow-up time of 441 days (IQR, 201.5).

Figure 3
Figure 3

Kaplan-Meier curve demonstrating the proportion of dogs with pulmonary coccidioidomycosis and radiographic evidence of TBL on baseline evaluation compared with length of time to radiographic TBL resolution. Each step down in the curve represents radiographic resolution of TBL in a dog with pulmonary coccidioidomycosis.

Citation: Journal of the American Veterinary Medical Association 261, 11; 10.2460/javma.23.04.0220

Variables associated with TBL presence and severity

The ordered logit model did not identify associations between presence or subjective severity classification of TBL with AGID IgM positivity (P = .66), AGID IgG positivity (P = .73), EIA IgG positivity (P = .38), or IgG inverse titer values (P = .35). Correlation was strongest between AGID IgM positivity and several semiobjective TBL size ratios, including height:LT4 (rPB = 0.49; 95% CI, 0.10 to 0.71) and height:LM (rPB = 0.49; 95% CI, 0.09 to 0.71; Table 1).

Table 1

Correlation between serologic antibody testing results and ratios of tracheobronchial lymph node area, height, and length compared with T4 and manubrial length in 26 dogs with pulmonary coccidioidomycosis diagnosed between October 2020 and February 2021. Point-biserial correlation expressed for relationship between variables and binary test results (positivity); Spearman rank correlation expressed for nonparametric numerical test results (inverse titers).

Variable AGID IgM positivity AGID IgG positivity EIA IgG positivity AGID IgG inverse titer
Area:T4 ratio 0.31 –0.05 0.14 –0.10
Area:manubrium ratio 0.38 –0.03 0.17 –0.23
Height:T4 ratio 0.49 –0.11 0.02 –0.03
Height:manubrium ratio 0.49 –0.23 –0.01 –0.22
Length:T4 ratio 0.28 –0.13 0.12 –0.12
Length:manubrium ratio 0.30 –0.27 0.14 –0.36

AGID = Agar gel immunodiffusion. EIA = Enzyme immunoassay. Ig = Immunoglobulin. T4 = Fourth thoracic vertebra length.

Variables associated with TBL resolution

Cox’s proportional hazard univariate analysis identified higher TBL severity classification (hazard ratio [HR] = 0.40; P = .02) and increasing TBL length:LT4 ratio (HR = 0.41; P = .01) as being associated with reduced probability of TBL resolution (ie, longer time to resolution; Table 2). When both of these variables were placed in a multivariable Cox model, the model was unstable, and neither variable (TBL subjective severity grade [HR = 0.49; P = .06] and TBL length:LT4 ratio [HR = 0.51; P = .06]) retained statistical significance.

Table 2

Associations between baseline variables and time to resolution of tracheobronchial lymphadenopathy in 25 dogs with pulmonary coccidioidomycosis using a univariate Cox proportional hazard model. Hazard ratios < 1 indicate reduced probability of resolution.

Variable HR 95% CI P value
AGID IgM positivity 0.94 0.38–2.30 .89
AGID IgG positivity 1.95 0.45–8.46 .37
EIA IgG positivity 0.94 0.41–2.17 .89
AGID IgG inverse titer 1.00 0.99–1.01 .88
Area:T4 ratio 1.00 0.99–1.00 .79
TBL grade 0.40 0.19–0.84 .02
Area:manubrium ratio 1.00 0.99–1.00 .77
Height:T4 ratio 0.55 0.24–1.26 .16
Height:manubrium ratio 0.39 0.05–3.00 .36
Length:T4 ratio 0.41 0.20–0.82 .01
Length:manubrium ratio 0.24 0.05–1.06 .06
Fluconazole dose 0.95 0.86–1.05 .31
Prednisone use 0.66 0.26–1.72 .40
Age 1.02 0.90–1.16 .72
Weight 1.01 0.97–1.04 .73

HR = Hazard ratio. TBL = Tracheobronchial lymphadenopathy.

See Table 1 for remainder of key.

Discussion

Pulmonary coccidioidomycosis can pose a diagnostic challenge in dogs, and information regarding expected temporal changes in radiographic TBL remains unclear. Therefore, this study aimed to determine whether any associations exist between serologic antibody test results and radiographic TBL in dogs with pulmonary coccidioidomycosis. Moreover, we sought to evaluate the time to resolution of radiographic TBL and whether any baseline variables were associated with this finding. We found that radiographic TBL presence and severity were not associated with antibody serologic test results. Resolution of TBL occurred in approximately 75% of dogs by the 3-month evaluation after initiation of fluconazole. Univariate analysis identified higher TBL severity and increasing TBL length:LT4 ratio as being associated with a longer time to resolution of TBL.

There were no clinically relevant associations between the presence or severity of radiographic TBL and antibody serologic test results in dogs with pulmonary coccidioidomycosis in the current study. To the authors’ knowledge, no other studies have specifically investigated the relationship between antibody serology and severity of radiographic TBL in pulmonary coccidioidomycosis in veterinary species. The lack of association in our study is important because it reinforces that clinicians should not make diagnostic decisions based on an assumed association between antibody serologic test results and radiographic TBL in dogs that have lived in or traveled to endemic regions. For example, pulmonary coccidioidomycosis should not be ruled out in dogs with radiographic TBL simply because they have negative or low antibody serologic test results. The reason for the lack of relationship between antibody serology and radiographic TBL in our study is likely multifactorial and beyond the scope of this study. One possible explanation is that the time course for anti–Coccidioides spp antibody kinetics and development of radiographically evident TBL may not occur in parallel. Several studies2022 in dogs with experimentally induced coccidioidomycosis highlight the variability in time to antibody seroconversion, maximum antibody levels, and development of radiographic TBL after inoculation. Another consideration is that there may be individual host immunologic factors that affect serologic antibody levels independent of the development of TBL.

Cox proportional hazards univariate analysis revealed that higher TBL severity classification and TBL length:LT4 ratios were associated with a longer time to resolution of TBL in dogs with pulmonary coccidioidomycosis in our study. The median time to resolution of radiographic TBL was approximately 3 months. Taken together, these results indicate that most dogs with pulmonary coccidioidomycosis will achieve resolution of radiographic TBL within 3 months of starting fluconazole, but larger tracheobronchial lymph nodes may take longer to resolve. Results from Shubitz et al21 show that fungal burden was associated with the severity of thoracic lymphadenopathy in dogs with experimentally induced coccidioidomycosis. Therefore, it is reasonable to surmise that dogs with naturally occurring pulmonary coccidioidomycosis and larger radiographic TBL take longer to resolve because they have a higher burden of disease. Alternatively or in addition, protracted lymphadenopathy could persist secondary to lymphatic obstruction.4

Another important finding from our study was that radiographic TBL did not develop in any dog if not present on baseline evaluation, and the severity of TBL did not worsen over its baseline status in any affected dogs over the observation period in this study, which concluded after dogs achieved clinical remission or a maximum of 12 months. Therefore, these results indicate that a dog with pulmonary coccidioidomycosis may have refractory or progressive disease and that a change in antifungal treatment should be considered if radiographic TBL develops or worsens during the course of treatment. Future studies are needed to confirm or refute this theory.

Our study had several limitations that must be considered. Dogs in this study were diagnosed with pulmonary coccidioidomycosis based on a collective interpretation of commonly used clinical criteria rather than identification of fungal organisms using cytologic, histologic, or microbiologic methods. Reaching a definitive diagnosis using these methods requires invasive and expensive diagnostic tests that are not typically pursued clinically. Another limitation is that radiologists were not blinded to the suspected diagnosis of pulmonary coccidioidomycosis at the baseline evaluation, possibly introducing bias into the determination of presence or absence of supportive radiographic findings. Next, the measurement of each individual right, left, and middle tracheobronchial lymph node is not possible because of radiographic superimposition, and thus we measured the entire increase in tracheobronchial soft tissue as a unit, likely reflecting a combination of all 3 lymph nodes. This approach may have led to an overestimation of size, particularly considering that only lateral radiographs were used for size determinations. Increased accuracy may be gained through use of thoracic CT23 in future studies, especially in cases where lymphadenopathy is mild or radiographically absent. This would also provide superior discrimination between perihilar soft tissue and superimposed ribs or vascular structures, mild left atrial enlargement, or a pulmonary lesion.24 However, CT evaluation is associated with increased cost and the need for sedation or general anesthesia, thus making it less likely to be a satisfactory screening and/or monitoring tool. Radiography is currently the predominant imaging modality used for preliminary diagnostic investigation in dogs with signs related to respiratory tract disease and is thus more clinically applicable. Eight dogs were administered anti-inflammatory doses of prednisone for a short duration after diagnosis, which may have affected lymph node size despite not being associated with time to resolution. It is unclear whether the dose or short duration of administration would have resulted in sustained improvements detected at the 3-month evaluation. The lack of association between fluconazole dose and time to resolution of radiographic TBL should be interpreted with caution. The delivered per os fluconazole dose can result in variable serum drug concentrations due to varied absorption and metabolism in dogs.25 Moreover, a recent study26 highlighted that not all FDA-approved generic formulations of fluconazole are bioequivalent in dogs, as they are in humans. Future studies should consider prioritizing utilization of serum fluconazole concentrations rather than administered doses when investigating various outcomes in dogs with coccidioidomycosis.

In conclusion, this study did not identify clinically relevant associations between the presence or severity of radiographic TBL and serologic antibody test results in dogs with pulmonary coccidioidomycosis, indicating that interpretation of these test results in tandem provides no added clinical value. Radiographic TBL was a common finding in this study and resolved in most dogs within 3 months after starting fluconazole. Univariate analysis found that higher TBL severity classification and TBL length:LT4 ratios were associated with a longer time to resolution of TBL. These results suggested that dogs with more severe radiographic TBL at the time of diagnosis have a higher likelihood of this imaging abnormality taking longer to resolve.

Supplementary Materials

Supplementary materials are posted online at the journal website: avmajournals.avma.org

Acknowledgments

Dr. Moore is a member of the Journal of the American Veterinary Medical Association (JAVMA) scientific review board. He declares that he had no role in the editorial direction of this manuscript.

Preliminary data from this study were presented at the virtual European Congress of Veterinary Internal Medicine for Companion Animals in 2021.

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