Diagnosis of invasive fungal infections in small animals

dvm360dvm360 July 2023
Volume 54
Issue 7
Pages: 63

Learn how to navigate challenges and make a speedier diagnosis

Hanzlicek is a veterinary internal medicine specialist with more than 14 years of professional experience teaching, providing clinical services, and conducting research in small animal internal medicine. He recently served for 4 years as the Joan Kirkpatrick Chair of Small Animal Medicine and director of the residency training program of small animal internal medicine at Oklahoma State University in Stillwater. He has lectured in the preclinical curriculum at multiple veterinary colleges and has provided continuing veterinary education around the world, discussing diagnosis, management, and prevention of small animal disease.

Win / stock.adobe.com

Win / stock.adobe.com

As supervisor of the Bioscience Research Laboratory, he has conducted research in the areas of infectious disease, hematology, and whole blood coagulation, and he has more than 40 publications to his credit. As a primary investigator and collaborator, he has received more than $1 million in grant-funded research. He has a strong interest in researching invasive fungal infections and has more than 15 publications directly related to endemic fungal infections. As veterinary services director at MiraVista Diagnostics, he oversees veterinary consultation and veterinary research studies and provides strategic leadership to expand MiraVista Diagnostics’ menu of veterinary services and products.

Invasive fungal infections (IFIs) are a diagnostic challenge. IFIs mimic other infectious, inflammatory, or neoplastic diseases. The lack of early clinical suspicion and other diagnostic challenges delay appropriate treatment. For example, it takes an average of 4 to 6 weeks after the start of signs before histoplasmosis is diagnosed in dogs and cats.1,2

Identification of organisms via pathology (cytopathology or histopathology) or culture is confirmatory, but both have limitations. Sampling of certain anatomic locations can be invasive and/or not feasible, including the lung, tracheobronchial lymph nodes, central nervous system, vertebrae, or eyes. Low numbers of organisms can be missed, especially on routine hematoxylin and eosin stains, and special stains (eg, Grocott methenamine silver and periodic acid-Schiff) should be requested for suspicious cases.

Culture of affected tissues can be insensitive for certain pathogens, even when organisms are seen on pathology. For example, the diagnostic sensitivity of tissue/fluid culture of dogs and cats with histoplasmosis was 52% and 79%, respectively, in one cohort.3 In addition, long turnaround time (up to 6 weeks) limits the clinical usefulness of fungal culture for certain pathogens. Because of these limitations, noninvasive biomarkers (antigens, antibodies) are often needed to support the diagnosis of IFI.

Antigen detection

Polysaccharides, which are continuously shed from the fungal cell wall, are the most common fungal antigen biomarkers. They have the advantage of being specific for active disease in contrast with serology, where low antibody levels can be caused by past exposure. In addition, polysaccharide antigens are very stable over a wide range of temperatures (freezing to boiling) and storage times (weeks refrigerated to indefinitely frozen). Certain antigens can be detected in urine, which is often abundant and simple to collect. Because antigens are specific for active disease, they are useful for treatment monitoring. Limitations of antigen biomarkers include cross-reactivity of closely related organisms as seen between Histoplasma and Blastomyces or Aspergillus and other hyaline molds.

Blastomyces, Coccidioides, and Histoplasma

Galactomannan (GM) detected by enzyme immunoassay (EIA), which is commercially available from MiraVista Diagnostics, is the antigen biomarker used to support the diagnosis of blastomycosis, coccidioidomycosis (valley fever; VF), and histoplasmosis. GM can be detected in body fluids including serum, urine, cerebrospinal fluid (CSF), and bronchoalveolar lavage fluid (BALf). Urine is preferred over serum because it generally contains a higher concentration of GM and thus provides a higher diagnostic sensitivity. In less than 5% of cases, urine antigen testing yields a negative result whereas serum antigen testing yields a positive result, necessitating the testing of both sample types. There is high cross-reactivity between Blastomyces and Histoplasma GM. Initial testing for both antigens on the same sample type is not cost-efficient. There is moderate cross-reactivity between Coccidioides GM and Blastomyces and Histoplasma GM.

The pooled diagnostic sensitivity (see Table) across findings from multiple studies of GM EIA testing with urine samples from cats and dogs with blastomycosis or histoplasmosis is 92% to 95%.4-11 Unfortunately, the diagnostic sensitivity of GM EIA for the diagnosis of VF is considerably lower. For example, testing both urine and serum for GM provided a sensitivity of 20% in one study.12 This is likely due to lower amounts of GM shed by Coccidioides. Unlike blastomycosis and histoplasmosis, antibody detection is the initial biomarker of choice for VF.

Table. Diagnostic Performance of Commercially Available Fungal/Oomycete Biomarker Tests in Cats and Dogs4-16, 20, 22, 24, 25, 27, 28, 31-40

EIA, enzyme immunoassay; GM, galactomannan; GXM, glucuronoxylomannan; ID, immunodiffusion; LA, latex agglutination; NA, not applicable. Bolded tests indicate primary biomarker. When available, pooled average from peer-reviewed publications was used.

Table. Diagnostic Performance of Commercially Available Fungal/Oomycete Biomarker Tests in Cats and Dogs4-16, 20, 22, 24, 25, 27, 28, 31-40

EIA, enzyme immunoassay; GM, galactomannan; GXM, glucuronoxylomannan; ID, immunodiffusion; LA, latex agglutination; NA, not applicable. Bolded tests indicate primary biomarker. When available, pooled average from peer-reviewed publications was used.


Glucuronoxylomannan (GXM) detected by latex cryptococcal agglutination test (LCAT) is the antigen biomarker of choice to support the diagnosis of cryptococcosis. Common sample types include serum and CSF. The assay includes latex beads coated with anti-GXM antibodies. If GXM is present in the sample, the beads clump together. If 50% or more of the beads are clumped at the lowest dilution (1:2), it is considered a positive result. Further dilution titers are then tested with the highest dilution, with 50% or more of the beads clumped together being the reported antigen titer. There are multiple manufacturers of LCAT materials, and even with the same manufacturer, there is high variability between laboratories regarding dilution titers. The positive vs negative result is generally consistent, however. If GXM titers are used for treatment monitoring, the same service laboratory should be used over time.

There is no cross-reactivity with GXM and GM used for blastomycosis, histoplasmosis, or VF. Occasionally, animals with cryptococcosis will yield a low level (0.5-1.5 IU) and false positive result with Aspergillus GM EIA (unpublished data). The opposite scenario, with aspergillosis causing a false positive result with Cryptococcus GXM LCAT, has not been documented. The pooled diagnostic sensitivity of GXM LCAT in findings from several published studies is 91% and 98% in dogs and cats, respectively, with a diagnostic specificity near 100%.13-16

FDA-cleared Cryptococcus GXM EIAs from IMMY and Meridian Bioscience are commercially available and intended for human testing. There are no published data regarding the diagnostic performance in veterinary species. There is a single case report of a cat with pathology-proven nasal cryptococcosis and multiple false negative EIA results, whereas LCAT results were clearly positive.17 Further research is needed before GXM EIA testing can be recommended in veterinary species. Likewise, an FDA-approved GXM lateral flow assay (LFA) from IMMY intended for near-patient testing use in humans is commercially available. This device is not approved by the United States Department of Agriculture (USDA) for use with veterinary species. Its diagnostic performance has not been described, but it has been compared with LCAT results. In 2 studies, the LFA yielded a positive result in 95% (35 of 37) of LCAT positive samples and negative result in 93% (241 of 259) of LCAT negative samples.18,19 The device has been recalled in the past because of unexpected false positive results. Depending on the clinical scenario, the decrease in turnaround time of approximately 24 to 48 hours may be beneficial. Even with a positive LFA result, the LCAT is still needed to obtain baseline GXM titer for treatment monitoring.


Aspergillus GM EIA was cleared by the FDA in 2002 (Platelia; Bio-Rad) to detect Aspergillus GM in human serum and support the diagnosis of invasive aspergillosis (IA). More recently, it was cleared to test human BALf. In humans, testing is often repeated multiple times while they are at high risk for IA with neutropenia, following organ transplant, or with other immunosuppressive conditions. An assay from MiraVista Diagnostics has been validated in dogs and cats to detect GM in serum, urine, CSF, or BALf. Findings from a study of dogs with culture-proven IA showed the diagnostic sensitivity to be 92% and 88% when serum and urine were tested, respectively.20 The diagnostic specificity was 86% and 92% for serum and urine, respectively.20 In that study, increasing the diagnostic cutoff from 0.5 IU (recommended by the manufacturer) to 1.5 IU increased specificity to 93% for both sample types without sacrificing sensitivity. When both serum and urine results were considered in parallel (either test result is positive), the sensitivity increased to 100%, suggesting that both sample types should be tested in some animals. If one sample type must be chosen, serum is the first choice. Interestingly, guidelines for the diagnosis of IA in humans have also recommended a higher diagnostic cutoff (1 IU for a single sample or 0.7 IU for serum and 0.8 IU for BALf) to improve specificity.21

Aspergillus GM EIA is inaccurate for sino-orbital or sinonasal aspergillosis (SNA), and anti-Aspergillus antibody detection is the biomarker of choice.22 GM of many hyaline molds is cross-reactive with Aspergillus GM, so a positive result is not specific for aspergillosis but is strongly suggestive of aspergillosis or related invasive mold infection. Penicillin antibiotics can potentially cause a low false positive result. Vetivex pHyLyte (Dechra) causes a high false positive result. It contains gluconate, which is produced by Aspergillus fermentation of carbohydrates (sorghum). During production, the gluconate is contaminated with Aspergillus GM. In the past, this was seen with Plasma-Lyte (Baxter) but is no longer an issue because the source of gluconate was changed in 2016.23 A minimum 72-hour washout after administration of penicillin antibiotics or Vetivex pHyLyte is recommended before samples are collected for Aspergillus GM EIA.


An FDA-cleared β-D-glucan (βDG) assay is commercially available (Fungitell; Associates of Cape Cod), intended for human serum testing. βDG is found in the cell wall of many pathogenic fungi and serves as a broad fungal biomarker. Many molds, including Aspergillus, Pneumocystis, Candida, Histoplasma, Blastomyces, Coccidioides, and even Pythium (oomycete) shed βDG, which can be detected in serum and potentially other body fluids. βDG testing does not provide the same sensitivity as species-specific biomarker testing, which is recommended before βDG testing. For example, if blastomycosis is suspected, testing urine for GM antigen and testing serum for IgG antibody is recommended before βDG testing. βDG testing is most useful for suspected invasive mold infections after Aspergillus GM EIA yields a negative result. It also provides the only serum-based biomarker for Pneumocystis pneumonia. The available assays currently use an enzyme in the coagulation cascade of horseshoe crabs. Assays using nonanimal-derived materials are currently being developed.

Antibody detection

Antifungal antibody detection assays are primarily either EIA or immunodiffusion (ID). In general, EIAs have a higher analytical and diagnostic sensitivity compared with ID.5,22,24,25 The only indications for fungal ID antibody testing are to support the diagnosis of VF or SNA. Because of superior diagnostic performance, EIA has replaced ID for antibody detection for Blastomyces and Histoplasma.5


There are several manufacturers of commercially available ID test materials or test kits, and these can be produced in house in some service laboratories. Patient serum is added to a well adjacent to fungal antigen and positive control serum in clear agarose gel. A visible line of precipitation between patient serum and fungal antigen is suggestive of antigen-antibody complexes. If the line joins the precipitation line of the control serum, this is considered a positive result. No visible line or a line that does not join with the positive control line is considered a negative result. Traditionally, Coccidioides ID uses 2 antigen preparations: tube precipitin (TP) and complement fixing (CF). Each antigen is tested separately, with the primary antibody response to TP being IgM and response to CF being IgG. If detected, dilution titers are determined for CF antibodies. Histoplasma ID includes 2 antigen preparations (H and M antigens). Aspergillus ID often includes culture-derived antigens from multiple Aspergillus species, and Blastomyces ID uses A antigen.

Enzyme immunoassay

EIAs for detection of antifungal IgG are commercially available for Coccidioides, Blastomyces, and Histoplasma in dogs and Histoplasma in cats (MiraVista Diagnostics). In addition, there are antibody detection EIAs available for Pythium, a fungal-like oomycete. As noted previously, the diagnostic performance of EIA is often superior to ID. EIA also has the advantages of improved clinical and laboratory efficiency, easier scalability, and shorter turnaround time. Available EIAs use proprietary fungal/oomycete antigens for antibody capture/detection.


Because of the lack of a sensitive Coccidioides antigen test, anti-Coccidioides antibodies are the primary biomarker used to support the diagnosis of VF. In one study, when assays were compared head to head, the EIA had a higher diagnostic sensitivity when compared with ID for pathology-proven cases (89% vs 73%).25 A second study using test materials from a different manufacturer had similar findings, with the EIA having a higher diagnostic sensitivity (93% vs 86%).26 Findings from several studies showed the pooled diagnostic sensitivity of ID is 87%.25-28 Either assay can detect antibodies in animals with VF when the other assay has a false negative result; thus, testing for antibodies in both assays might be needed. In one study, performing both assays increased the diagnostic sensitivity to 95.5% in pathology-proven cases and 98.6% when probable cases were also included.25

An anti-Coccidioides antibody LFA (IMMY) for near-patient testing has been reported in dogs.26,29 It is not FDA cleared for human testing and is not USDA approved for veterinary testing. The LFA yields a negative result in 11% to 14% of samples that yield positive results with EIA or ID and yields positive results in 15% to 20% of samples that yield negative results with EIA or ID.26,29 For this reason, a positive or negative LFA result should be confirmed with EIA or ID, which are also needed for baseline antibody levels for treatment monitoring.


Anti-Aspergillus antibodies are the preferred biomarker for sino-orbital aspergillosis or SNA in cats and dogs. In the United States, anti-Aspergillus antibody assays are currently ID. The presence of ID antibodies, along with consistent clinical findings, is highly specific (nearly 100%) for SNA.22,24 The diagnostic sensitivity is 77% and 43% in dogs and cats, respectively, and a negative test result does not rule out SNA.22,24 EIAs reported in the literature have a higher sensitivity but slightly lower specificity when compared with ID.22,24 Currently, EIAs are not commercially available in the United States.


Anti-Histoplasma antibody detection via ID has an unacceptably low diagnostic sensitivity (0%-14%) and is not recommended in cats or dogs (unpublished data). In one cohort, IgG EIAs provided a diagnostic sensitivity of 81% and 77% in cats and dogs, respectively. Diagnostic specificity of IgG EIAs was 95% and 93% in cats and dogs, respectively. Most importantly, IgG EIA yields a positive result in animals with histoplasmosis who attain a false negative result from a GM antigen test. For this reason, IgG EIA is most useful when GM testing of urine yields a negative result and suspicion for histoplasmosis remains.


Anti-Blastomyces antibody detection via ID has an unacceptably low diagnostic sensitivity and is not recommended in dogs.5,6 It is currently the only available anti-Blastomyces antibody test in cats. A commercially available anti-Blastomyces IgG EIA (MiraVista Diagnostics) uses BAD1 antigen for IgG capture/detection. BAD1 antigen is only expressed after Blastomyces converts from mold in the environment to yeast in the body. In one study, the diagnostic sensitivity and specificity in dogs was 95% and 96%, respectively. IgG EIA should be considered when GM antigen testing of urine yields a negative result and suspicion of blastomycosis remains.


Anti-Pythium IgG EIA is the biomarker of choice for pythiosis. The diagnostic performance has not been reported from all service laboratories that offer this assay. In certain service laboratories, it is highly accurate, providing a diagnostic sensitivity and specificity greater than 98% in dogs and cats with pythiosis (unpublished data).30 It should not be assumed that all anti-Pythium antibody tests offered commercially have similar diagnostic performances. The high diagnostic performance of some assays is in part due to the chronic course of disease and the resultant robust humoral immune response. In addition, Pythium is immunogenically different from most fungal pathogens, and cross-reactivity is uncommon. Cross-reactivity with rare causes of disease—zygomycetes and other oomycetes (Paralagenidium and Lagenidium)—in veterinary species is possible.

DNA detection

Nucleic acid amplification tests (NAATs), most commonly real-time quantitative polymerase chain reaction (qPCR), are commercially available for fungal DNA detection. The diagnostic performance and clinical utility of these tests are mostly unknown in veterinary species. Because of the high diagnostic performance of antigen and antibody detection tests, which use convenient sample types (whole blood, serum, urine), it is unlikely that NAATs will ever be the primary fungal biomarker. NAATs might be most useful when testing a small sample volume (CSF, aqueous, BALf, joint fluid) for multiple pathogens, which is possible with multiplex assays. Additional research is needed to better understand the clinical utility of currently available qPCR assays. Studies should include various sample types from animals with naturally occurring IFI. Pan-fungal PCR and sequencing can provide fungal speciation when organisms are seen on cytology slides or in formalin-fixed paraffin-embedded tissues. This is most useful for hyphae, where morphology is similar across many fungal/oomycete species.

Combined biomarker testing

Because of the challenges of diagnosing IFI, testing for multiple biomarkers for the same fungal pathogen might be indicated. As previously discussed, this could include testing serum and urine for Aspergillus GM, urine for Histoplasma or Blastomyces GM, and serum for IgG or anti-Coccidioides antibodies via EIA and ID. In addition, because of overlapping clinical findings and endemic geographic ranges, biomarker testing for multiple fungal pathogens in the same animal might be needed.


Diagnosing IFI can be challenging. Necessary testing to support the diagnosis might include pathology, fungal culture, and biomarker testing. Early clinical suspicion and appropriate biomarker test and sample selection can decrease time to diagnosis and appropriate treatment, potentially improving outcomes.


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