Coccidioides posadasii and Coccidioides immitis are soil-dwelling fungi found in the western United States, Mexico, and Central and South America that cause coccidioidomycosis, otherwise known as Valley fever (VF).1–4 While Coccidioides spp infect various mammals aside from humans, exposure is common in dogs that travel to or live in endemic regions.5,6 In 1 study5 that examined dogs raised in Pima and Maricopa counties in Arizona, it was estimated that 11% and 28% of dogs would become infected with Coccidioides spp by 1 and 2 years of age, respectively. Following infection, affected dogs may develop clinical signs associated with acute to chronic respiratory tract disease along with nonspecific signs of systemic illness with the potential for dissemination to various sites including the skin, bone, and CNS.6
Rapid and accurate detection of infection with Coccidioides spp is important in initiating appropriate antifungal treatment. Definitive diagnosis is achieved using organism-based methods including cytology, histopathology, and culture; however, specimen retrieval is often invasive, and organism identification in tracheal washes and bronchoalveolar fluid is infrequent.7–9 Antigen-based methods such as PCR and detection of Coccidioides galactomannan by enzyme immunoassay (EIA) have been reported, but are insensitive and infrequently used in dogs.6,10–12 Thus, the initial diagnosis of VF in dogs relies on clinical presentation and serology. Agar gel immunodiffusion (often referred to as AGID or ID) is traditionally used for the demonstration of anti-coccidioidal IgM and IgG antibodies and can be performed quantitatively to measure an antibody titer. Chitinase 1 (CTS1) is the seroreactive component in ID antigen preparations for detection of anti-coccidioidal IgG in humans,13–16 although the same reagents are employed for ID regardless of species. Detection of anti-coccidioidal antibodies cannot distinguish exposure from active disease, as antibody titers between ≤ 1:2 and 1:16 have been demonstrated for both clinically and sub-clinically infected dogs.5,17 Antibody titers ≥ 1:32 are suggestive of a higher likelihood of disseminated disease, although there are instances of dogs with disseminated disease with negative or lower (< 1:32) IgG titers.18–20 Although a single titer is not a reliable indicator of disease status or severity on its own,5,17 antibody detection remains an important aid for contextualizing the likelihood of disease in combination with other diagnostic testing.8
The use of ID for detecting anti-coccidioidal IgG antibodies in dogs with diagnosed or proven coccidioidomycosis has been demonstrated to be 94% to 100% specific and 73% to 86% sensitive.21–23 Although specific, the suboptimal sensitivity of ID combined with its turnaround time of 3 to 7 days24 has led investigators to explore more rapid methods such as antibody EIAs and lateral flow assays (LFAs). While commercial Coccidioides antibody EIAs are available, their use of antihuman secondary antibodies requires modification to be used with dog sera. In contrast, 2 Coccidioides antibody LFAs that have been developed are species agnostic and can be performed with dog sera without any adjustment to the test. One LFA is the sōna Coccidioides antibody LFA (IMMY), which detects both IgM and IgG antibodies against a proprietary and undefined mixture of coccidioidal antigens adsorbed to a nitrocellulose strip. A result can be interpreted 30 minutes after the test is set up, and preliminary studies24,25 with dog sera have reported a positive percent agreement of 87.5% to 88.9% and negative percent agreement of 85.2% to 100% when compared to ID. The other LFA was recently developed by some of the authors of this paper (FJG, TEG, and DFL) that detects antibodies against coccidioidal CTS1 in 10 minutes.26 The accumulation of antibody-CTS1 antigen complexes at the test line is measured by an LFA reader that provides an objective numerical result that was previously shown to positively correlate with complement fixation antibody titers in humans.26 To further explore the utility of LFAs, here we evaluated the performance of both the sōna and CTS1 LFAs in comparison to ID IgG results in 73 dogs with coccidioidomycosis.
Methods
Sample selection
Stored sera from client-owned dogs diagnosed with coccidioidomycosis were evaluated retrospectively. Serum specimens evaluated in the current study were originally procured from dogs enrolled in various other studies that had sufficient surplus sera available or surplus sera from other diagnostic investigations from dogs evaluated at the Midwestern University College of Veterinary Medicine. Only 1 specimen from each dog was used. Dogs were excluded if after a review of the medical record, there was insufficient evidence to make a highly probable coccidioidomycosis diagnosis made by a single board-certified small animal internist (JAJ). Serum specimens were collected at the initial screening evaluation for coccidioidomycosis and were stored at –80 °C for a maximum of 4 years.
Criteria for selection of cases
A diagnosis of coccidioidomycosis was made using a collective interpretation of clinical findings, available diagnostic test results, and detection of serum anti-coccidioidal antibodies. Serum antibodies against Coccidioides spp were detected at commercial laboratories by either ID (Antech Diagnostics, IDEXX Laboratories, MiraVista Labs, and Protatek Reference Laboratory) or EIA (MiraVista Labs). Dogs were required to have at least 1 positive anti-coccidioidal serological test result (ie, ID IgM, ID IgG, or EIA IgG). Dogs were categorized as having either pulmonary or disseminated disease. Dogs with pulmonary coccidioidomycosis were required to have at least 1 clinical sign associated with respiratory tract disease in conjunction with thoracic imaging abnormalities. Dogs with unremarkable thoracic imaging results were included if there was clinical or serological improvement with antifungal treatment. Dogs with disseminated coccidioidomycosis had confirmation or a high index of clinical suspicion of disease in 1 or more organs outside of the thoracic cavity.
Agar-gel immunodiffusion
Since ID testing was performed by various different diagnostic veterinary commercial laboratories at the time of original diagnosis, ID and quantitative ID (determination of antibody titers by ID) were performed using a single uniform ID reagent on all dog sera as previously detailed.26 Briefly, 20 µL of sera was tested against 20 µL of Coccidioides antigen in a standard 7-well ID pattern and incubated for 48 hours in a humidified chamber at 27 °C. The presence or absence of a line of precipitin between the patient and antigen wells was recorded and any dog with a positive result was run by quantitative ID, whereby the dilution of serum at which reactivity was last seen was reported as the serum antibody titer. The maximum dilution tested was 1:256.
Coccidioides spp antibody LFAs
Each dog serum specimen was run on 2 different LFAs. The CTS1 Coccidioides antibody LFA was performed as previously described.26 Briefly, 6.8 µL of serum was added to the sample port followed by 2 drops (approx 60 µL) of chase buffer. The test line density was read after 10 minutes using an iDetekt RDS-2500 density reader (Detekt Biomedical). A density unit value ≥ 30,000 was considered a positive result, and a value < 30,000 was recorded as negative. A schematic of the CTS1 Antibody LFA workflow is illustrated (Figure 1). Precision testing was performed on a single lot of test strips using sera from 1 ID IgG-negative and 1 ID IgG-positive dog in replicates of 5 and calculating the coefficient of variation (CV) as a percentage as follows: %CV = (SD/mean) X 100%.
Schematic of chitinase 1 (CTS1) antibody lateral flow assay (LFA) and sōna antibody LFA. A—In the CTS1 LFA, serum is added to the sample port followed by chase buffer. The test is allowed to incubate for 10 minutes and is subsequently read using an iDetekt LFA reader. A red test line indicates a positive result, which is measured using the LFA reader. B—For the sōna LFA, 100 µL of a 1:441 dilution of serum is transferred to a flat-bottom tube or well. A sōna strip is inserted and interpreted visually after 30 minutes, with a red test line indicative of a positive result. This schematic was modeled from the manufacturer’s instructions and created using Biorender.com. C—Examples of each test are shown for 4 dogs with the result for each LFA designated as a “+” or “−” above the test strip; the CTS1 LFA density unit results are also shown above the cassette. For the CTS1 LFA, a test line density unit value < 30,000 is negative, while a value ≥ 30,000 is positive. Dog 57 was serologically negative by immunodiffusion (ID) and enzyme immunoassay and produced a negative result in the sōna (left) and CTS1 (right) LFA (test line density unit value, 12,590). Dog 56 was serologically positive by ID, with an antibody titer of 1:8 to 1:16 and positive on both the sōna and CTS1 LFAs (test line density unit value, 404,386). Dogs 71 and 72 were both serologically positive by ID with antibody titers ≥ 1:128 and were positive by both LFAs.
Citation: American Journal of Veterinary Research 85, 6; 10.2460/ajvr.23.12.0272
The sōna Coccidioides Antibody LFA (IMMY) was performed as detailed in the manufacturer’s instructions, which first required the dilution of sera 441-fold (10 µL serum added to 200 µL diluent and then 10 µL of the first dilution added to another 200 µL diluent). A sōna test strip was inserted into a flat-bottom well of a 96-well plate containing 100 µL of the diluted serum and incubated for 30 minutes. Results were interpreted by 2 independent observers according to the manufacturer’s instructions. A schematic of the sōna LFA workflow is illustrated (Figure 1).
Statistical analysis
The data were examined 2 different ways. First, we calculated the sensitivity with 95% CI for each test under the assumption that all dogs were infected at the time of serum collection (100% prevalence). The specificity, positive predictive value, and negative predictive value could not be calculated under this assumption since false positive results would not be possible. Next, we compared the results of each LFA to ID results and calculated their positive percent agreement (PPA) with 95% CI, under the assumption that ID IgG results were 100% accurate and therefore the reference standard for antibody detection. Specimens that returned an ID IgG result different from that reported by the commercial diagnostic lab that performed ID on a fresh specimen were excluded from this analysis. Specificity and PPA analyses were done using VassarStats Website for Statistical Computation. Spearman nonparametric rank correlation (rs) analysis was used to evaluate the correlation between in-house ID IgG titer and CTS1 LFA test line density units. Correlation coefficient values were interpreted as follows: 0 to 0.09 negligible correlation; 0.10 to 0.39 weak correlation; 0.40 to 0.69 moderate correlation; 0.70 to 0.89 strong correlation; and 0.90 to 1.00 very strong correlation.27 Simple linear regression analysis was also performed to evaluate the relationship between in-house ID IgG antibody titers and test line density units of the CTS1 LFA. The ID IgG titer values were log-transformed to meet the assumptions for linear regression (ie, normally distributed data and homoscedasticity of residuals). Analyses were performed using GraphPad Prism v10.0.3. A P value less than .05 was considered significant.
Results
Seventy-three dogs were included in this study. The median weight was 21.6 kg (IQR, 11.6 to 30.5), and the median age was 5.5 years (IQR, 2.1 to 8.4). There were 51 purebred dogs and 22 mixed-breed dogs. The most commonly represented purebred breeds included Labrador Retriever (n = 6), Pit Bull Terrier (5), German Shepherd Dog (4), beagle (3), and Chihuahua (n = 3). The sex distribution was as follows: castrated male (n = 36 [49%]), spayed female (25 [34%]), intact male (8 [11%]), and intact female (4 [6%]). One of the 73 dogs was included despite negative antibody serologic testing results because of microscopic confirmation of Coccidioides spp organisms. This dog had an appendicular bone lesion identified radiographically with cytological confirmation of Coccidioides spp spherules. Fifty (68%) dogs had a diagnosis of pulmonary coccidioidomycosis, and the remaining 23 (32%) dogs had disseminated disease. Dogs with pulmonary coccidioidomycosis had 1 or more improvements in clinical status, thoracic radiographic findings, or antibody serologic test results after initiation of antifungal therapy. Eight (33%) dogs with disseminated disease had identification of Coccidioides spp organisms on cytological or histopathological examination or isolated via fungal culture. Bone was the most common site of dissemination being found in 70% (16/23) of dogs confirmed with organism identification or visualization of 1 or more radiographic bone lesions (lytic, proliferative, or both). Two dogs were diagnosed with pericardial coccidioidomycosis based on clinical status, echocardiographic findings, and antibody serologic test results. Two dogs were suspected to have ophthalmic involvement. One dog was evaluated on an emergency basis and on abdominal ultrasound examination had enlarged intrabdominal lymph nodes, nodules embedded within the peritoneal wall, free peritoneal effusion, and nodules within the spleen. This dog’s AGID results were IgM (positive) and IgG ≥ 1:128. This dog was euthanized without a necropsy. Fifteen of the 16 dogs without confirmation of Coccidioides spp organisms had follow-up information that confirmed clinical, radiographic, echocardiographic, or antibody serologic improvement.
We first assessed the sensitivity of each serologic test (reference laboratory ID IgG, in-house laboratory ID IgG, CTS1 LFA, and sōna LFA) under the assumption of a prevalence of 100%. The sensitivity of all 4 methods ranged from 83.6% to 89.0% (Table 1). All 4 tests were positive for 52/73 dogs, and all 4 tests were negative for 2/73 dogs. The remaining 19 dogs had varying results for each test (individual test results for each dog are provided; Supplementary Figure S1).
Sensitivity of 4 different anti-Coccidioides antibody tests in 73 dogs with pulmonary or disseminated coccidioidomycosis, assuming 100% prevalence.
| Assay | Prevalence % | Sensitivity (95% CI) |
|---|---|---|
| Commercial ID (IgG) | 100 | 87.7% (77.4%–93.9%) |
| In-house ID (IgG) | 100 | 83.6% (72.7%–90.9%) |
| CTS1 LFA | 100 | 89.0% (79.0%–94.8%) |
| sōna LFA | 100 | 86.3% (75.8%–92.9%) |
CTS1 = Chitinase 1. ID = Immunodiffusion. IgG = Immunoglobulin G. LFA = Lateral flow assay.
The ID IgG results from 64 of 73 (88%) dogs agreed between our lab and those performed in other commercial laboratories. Of the 9 dogs with conflicting results, 3 were IgG positive and 6 were IgG negative when tested in our laboratory (Supplementary Figure S1). Because the cause of the discrepancy in ID results is not known, these 9 dogs were excluded when evaluating the PPA of each LFA against ID IgG results for antibody detection.
When comparing the CTS1 and sōna LFAs for detection of anti-coccidioidal antibodies in 64 dogs with corroborated ID results (ie, commercial laboratory and in-house testing), antibodies were detected by both LFAs in 53 of the 58 ID IgG-positive dogs. The other 5 sera that were positive for IgG by ID were positive on the CTS1 LFA but not the sōna LFA (Supplementary Figure S1). Of the 6 dogs that were negative for IgG by ID, 2 dogs did not have detectable antibodies by the CTS1 LFA or sōna LFA, but both LFAs detected antibodies in 1 IgG ID-negative dog. Additionally, sōna was positive for 3 dogs negative for IgG by ID, and upon a chart review, 2 of the 3 dogs were positive for IgM by ID at the baseline diagnosis with testing performed at commercial laboratories. Using ID IgG as the comparator for antibody detection, the CTS1 LFA showed a PPA of 100% (95% CI, 92.3% to 100%), and the sōna LFA showed a PPA of 91.4% (95% CI, 80.3% to 96.8%).
Because the CTS1 LFA has a measurable output (test line density units), we compared test line density values to in-house ID titers for the 58 ID IgG-positive dogs. Test line density units obtained for these dogs in relation to ID IgG antibody titer are shown (Figure 2) with an overall positive correlation observed. Spearman rank correlation and simple linear regression analysis of log2 ID IgG titer and CTS1 LFA density units resulted in a strong rank correlation coefficient (rs = 0.82; 95% CI, 0.71 to 0.89; P < .0001) and a coefficient of determination that describes 68% of the relationship between ID IgG antibody titer and CTS1 LFA test line density units (r2 = 0.68; P < .0001), respectively. Precision testing showed a test line density unit CV of ∼15% in an IgG ID-negative dog and ∼8% in an IgG ID-positive dog.
A—Relationship of density units measured by the chitinase 1 (CTS1) lateral flow assay (LFA) to in-house immunodiffusion (ID) antibody titers for 58 dog sera. All 58 dogs with a positive antibody titer by ID and their corresponding density unit values on the CTS1 LFA are graphed by antibody titer group. B—The horizontal line in each titer group represents the mean density unit value. Simple linear regression analysis and Spearman rank correlation (rs) are illustrated. The dotted lines represent the 95% CIs for the slope of the regression line.
Citation: American Journal of Veterinary Research 85, 6; 10.2460/ajvr.23.12.0272
Discussion
Clinical diagnosis of VF in dogs typically depends on a collective interpretation of clinical signs, clinicopathologic findings, diagnostic imaging, and antibody serologic testing, which detects IgM and IgG antibodies against coccidioidal antigens. Although the percentage of dogs started on antibiotics or other inappropriate treatments before a diagnosis of coccidiomycosis is unknown, a rapid point-of-care test would allow treatment decisions to be considered in real-time while the owner and dog are present in the veterinary clinic. Here, the performance of 2 LFAs designed for detection of anti-coccidioidal antibodies was evaluated.
When including all 73 dogs tested, anti-coccidioidal antibodies were detected by the CTS1 LFA in 65 (87.8%) dogs and by the sōna LFA in 63 (85.1%) dogs (Table 1). Thus, both tests had similar sensitivities in the context of this study, which included only dogs with clinical coccidioidomycosis. Although neither test differentiates whether IgM or IgG antibodies are detected at the test line, we elected to further compare the results of each test against ID IgG results. The ID IgM results were not initially considered because dogs solely positive for IgM are infrequent and are interpreted differently depending on other factors such as clinical signs and persistence over time.5,17 Using ID IgG results as a reference standard for antibody detection, the CTS1 LFA had a PPA of 100% while the sōna LFA had a PPA of 91.4%. Only 6 of 64 dogs were negative for IgG by ID. We went back and further reviewed the ID IgM results of these 6 negative dogs and found 3 to be positive for IgM by ID, 2 positive for IgG by MiraVista EIA, and 1 negative by all serologic methods. This provided an ID IgM-only occurrence rate of 4% (3/73 dogs) in our cohort, which did not include longitudinal monitoring of antibody response.
Other studies have compared the performance of the sōna LFA against ID.24,25 While the sōna LFA is relatively easy to use, its time-to-result is 3 times longer than the CTS1 LFA, there are more technical steps involved (dilution of sera 1:441), more consumables not provided with the test are required (tubes for dilution and flat-bottom tubes or plate for strip incubation), and the result is subject to observer interpretation. Although the sōna LFA was independently interpreted by 2 observers and no discordant results were reported, the interpretation may be prone to bias, especially in the current study, which included solely dogs with a clinical diagnosis of coccidioidomycosis. The CTS1 LFA is a more traditional approach to the LFA format, whereby patient serum or plasma is added directly to the cassette sample port that houses the LFA strip followed by 2 drops of chase buffer (Figure 1). The CTS1 LFA can be interpreted by the eye using a scorecard or can be read by an LFA reader as was done in this study to produce an objective, numerical result. We envision that this CTS1 LFA could be used in a veterinary clinic setting while the dog and owner are present so that treatment decisions can be made during the healthcare visit. Having a semiquantitative result at point-of-care has the potential to help clinicians contextualize the probability of an active clinical Coccidioides spp infection or pursue additional diagnostic tests. Additionally, a semiquantitative result that can estimate the IgG titer has the potential to be useful in monitoring treatment strategies as antibody serology data are an important component in conjunction with clinical status and diagnostic imaging when determining clinical remission. Studies, both prospective and longitudinal, are needed to properly evaluate the utility and accuracy of the semiquantitative feature of the CTS1 LFA.
While the objective of this study was to evaluate 2 different LFAs in dogs with coccidioidomycosis, this study has several limitations. First, our study was limited to dogs residing in 1 endemic region, and only specimens from dogs with coccidioidomycosis were selected resulting in an artificially high prevalence (100%). By assuming 100% prevalence, even the currently accepted standard for antibody detection (ID) resulted in < 100% sensitivity (Table 1). Second, no control group was used. These limitations prevented the evaluation of specificity, positive predictive value, and negative predictive value of each test on its own. However, we previously reported26 the performance of the CTS1 LFA using ID-positive endemic (n = 14) and ID-negative nonendemic (21) dog serum and showed a significant difference in test line density unit values between these groups. Definitive diagnosis of coccidioidomycosis and confirmation of the extent of disease requires identification of Coccidioides spp organisms via cytology, histopathology, or fungal cultures. This is not commonly accomplished in client-owned dogs with naturally occurring coccidioidomycosis. Instead, clinical diagnoses are generally made based on a collective interpretation of history, physical examination, hematology panel, serum biochemistry, imaging findings, and serologic test results. Therefore, it is possible, albeit unlikely, that some of the dogs included in this study did not have active clinical coccidioidomycosis. Moreover, it is possible that dissemination was missed in some of the dogs classified as having the pulmonary form of disease.
Additionally, our evaluation in this study was limited to determining sensitivity of each LFA in detecting anti-coccidioidal antibodies using ID results. The data were analyzed using ID IgG results from our laboratory using a uniform set of reagents to test all dog sera contemporaneously. This is a strength of our study because it is known that ID results are variable between laboratories.24 Some reasons for the discrepancies reported between our laboratory and commercial laboratories could be due to the test reagents used and protocol. For example, we do not concentrate serum, but it is possible that serum concentration is practiced in some commercial laboratories. We also quantify the amount of immunoreactive CTS1 in our ID antigen preparation using a previously reported EIA for standardization,28 while standardization of commercial reagents is not well described or understood. To remove these potential biases, it would be beneficial to evaluate both LFAs for their ability to detect acute coccidioidomycosis in veterinary hospitals where true prevalence is represented, and the same laboratory is utilized for ID results.
Without an accurate antigen-detection assay, coccidioidomycosis continues to be challenging to diagnose, especially when the currently accepted standard of antibody detection cannot differentiate between subclinical, active, and resolving infection. The use of antibody-based methods in conjunction with diagnostic imaging (when applicable) and clinical signs remains the current standard. However, the use of an LFA that can provide a comparable result to ID in 10 minutes versus 3 to 7 days may shorten the time frame for clinicians to start antifungal therapy in acutely sick dogs. Both LFAs evaluated here show good agreement with ID IgG results and similarly good to excellent sensitivities for a clinical diagnosis. While both can be interpreted by the eye, the CTS1 LFA may be further useful by producing a semiquantitative output.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org
Acknowledgments
The authors thank Lauren Chittick for her laboratory assistance. MiraVista Diagnostics provided some antibody serology testing for this study. The datasets generated are available upon reasonable request to the authors.
Disclosures
FJG, TEG, and DFL are inventors on an unlicensed patent application involving the CTS1 LFA evaluated in this report. TEG and DFL are cofounders of Cactus Bio, LLC. FJG is an employee of Cactus Bio, LLC. FJG, TEG, and DFL are part owners of Cactus Bio, LLC. All other authors have nothing to disclose.
No AI-assisted technologies were used in the generation of this manuscript.
Funding
Funding to support this work was provided by grants from the Flinn Foundation (grant No. 23-06534; to DFL), NIH (Small Business Innovation Research grant No. 1R43AI179331; to FJG and DFL), and Mayo Clinic Arizona DLMP Research Funding (D1-118; to TEG).
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