Introduction

Histoplasma capsulatum sensu lato, the causative agent of histoplasmosis, is a global saprophytic, dimorphic fungus that thrives in soil and bird and bat guano.^1,2 Inhalation of the microconidia from the mycelial form can result in a severe systemic mycosis affecting cats, dogs, people, and many other domestic and wildlife species.^1–4 Histoplasma spp are endemic to the central and eastern US and are extensively found in the Ohio, Mississippi, and St. Lawrence River valleys, although histoplasmosis has been diagnosed in nonendemic areas in humans and cats without a history of travel.^1,2,5,6

Histoplasmosis is reported to be the second most common fungal infection in cats and has a roughly 33% mortality rate within the first 6 months of diagnosis with appropriate antifungal therapy.^4,7 At the time of diagnosis, cats typically have disseminated disease, although clinical signs are often nonspecific.⁷ Lethargy, hyporexia, and weight loss have been historically noted in 67% to 85% of cats and respiratory signs in roughly 39% to 93% of cats.^7–9 Direct visualization of the organism, either by cytology or histopathology, confirms infection and renders additional testing unnecessary for a diagnosis.⁴ Aspiration of peripheral lymph nodes, when infiltrated with Histoplasma organisms, is minimally invasive, generally does not require chemical restraint, and provides a rapid and relatively inexpensive method of diagnosis. However, aspiration of lung, bone marrow, or intra-abdominal organs (such as spleen, liver, or abdominal lymph nodes) to establish a diagnosis of histoplasmosis is more invasive, may require chemical restraint, carries an increased risk of complications, and is technically more demanding than peripheral lymph node aspiration.

When clinical signs and imaging findings are compatible with histoplasmosis but collection of cytological or histological samples is considered too invasive, or if organisms are not found, antigen detection is often relied on to confirm a diagnosis of histoplasmosis in cats.^10–12 Quantification of the Histoplasma antigen in the urine by enzyme immunoassay (UA-EIA) is minimally invasive and has a reported sensitivity of 94% and specificity of 97% in cats with disseminated disease.¹¹ Alternatively, serologic detection of anti–Histoplasma immunoglobulin G antibodies by enzyme immunoassay (IgG-EIA) can also support a diagnosis of histoplasmosis in cats and has a reported sensitivity and specificity of 81.1% and 94.7%, respectively.¹³

Detection of Histoplasma DNA in biological samples may confirm a diagnosis of histoplasmosis in the absence of cytological or histological identification but is not routinely performed.⁴ The collection of blood from a peripheral vein for a CBC is routinely performed in the diagnostic evaluation of systemically ill cats, and this EDTA-anticoagulated whole blood provides a potential source for the detection of Histoplasma DNA in cats with disseminated histoplasmosis. In humans, whole blood RT-PCR has a reported sensitivity of 18% to 97% for the diagnosis of disseminated histoplasmosis, depending on whether a single or multiple blood samples are assayed per patient.^14–16 Although whole blood Histoplasma RT-PCR is commercially available for cats, there are no published data on the diagnostic performance. The guidelines for this test state that 2 mL of EDTA-anticoagulated whole blood is the ideal sample for systemic, nonspecific manifestations of histoplasmosis and that a negative result should prompt investigation of other clinical diseases, with the caveat that a negative PCR test result may be seen for various reasons.¹⁷

The objective of this study was to determine the diagnostic sensitivity and specificity of a commercial RT-PCR assay on EDTA-anticoagulated whole blood for the diagnosis of histoplasmosis in cats. A secondary aim of this study was to compare the sensitivity and specificity of the RT-PCR to IgG-EIA and UA-EIA for all cats enrolled in the study. The hypothesis was that the RT-PCR would have a sensitivity of approximately 70% and outperform IgG-EIA in accuracy.

Methods

Results

There were 21 cats in which the diagnosis was obtained cytologically or the attending veterinarian intended to submit tests for the diagnosis of histoplasmosis. Twenty cats met the inclusion criteria for the study, including 10 cats with histoplasmosis and 10 control cats. One cat diagnosed with histoplasmosis on cytology was excluded from the study because not all samples were collected. There were 19 cats from Kansas and 1 cat from Nebraska. The median age of all cats was 10 years (range, 1.5 to 15 years). The most common breed was domestic shorthair (n = 14). Other breeds included domestic longhair (n = 2), domestic medium hair (1), Maine Coon (1), Siamese (1), and Sphynx (1). There were 12 neutered male cats and 8 female cats (7 neutered).

Ten cats were diagnosed with histoplasmosis, including 6 cats with a proven and 4 with a probable diagnosis. These 10 cats ranged in age from 2 to 14 years (median, 10 years), and there were 7 neutered male and 3 neutered female cats. Cytological confirmation of histoplasmosis was obtained in 6 cats from aspirates of popliteal lymph node (n = 3), abdominal lymph node (2), spleen (1), liver (1), or gingival mass (1), as well as on examination of a peripheral blood smear (1). Organisms were identified in > 1 site in 2 cats. All cats were determined to have systemic histoplasmosis, although 3 cats presented with a history of chronic diarrhea and were suspected to have intestinal histoplasmosis as the primary site of infection. Two cats had been treated long-term (> 2 years) with prednisolone, one for plasma cell pododermatitis and the other for small cell intestinal lymphoma. One cat was reported to have tested positive for antibodies to FIV in the past and had also been treated with prednisolone for 3 months for undiagnosed pruritis immediately prior to the diagnosis of histoplasmosis. One additional cat had been receiving prednisolone for 5 weeks for presumed immune-mediated hemolytic anemia. Two cats had previously been treated for systemic histoplasmosis, had been in remission based on ≥ 2 negative UA-EIA results, and had not received antifungals for 8 and 25 months.

Anemia with associated weakness was the presenting complaint in 3 cats, with Hct ranging from 15% to 18% (reference interval, 35% to 50%) on presentation. Diarrhea with hyporexia was the main concern in 3 cats and evidence of systemic disease was identified in all 3. The clinical signs that prompted presentation to the Kansas State University Veterinary Health Center in the remaining cats included ocular disease, polyuria and polydipsia, dyspnea, and nasal congestion in 1 cat each. Thoracic radiographs were obtained in 4 cats, and diffuse interstitial to bronchointerstitial disease was identified in 3 cats. The 2 cats with relapsing disease had previously documented diffuse interstitial lung disease and were not radiographed. Leukocytosis was only present in 2 cats (total WBCs of 21,000 cells/µL and 48,400 cells/µL; reference interval, 4,200 to 19,100 cells/µL), both of which presented for chronic diarrhea. Monocytosis was present and mild in 5 cats, ranging from 500 to 700 monocytes/µL (reference interval, 0 to 500 monocytes/µL). Hyperglobulinemia was present in only 3 cats (range, 5.0 to 6.5 g/dL; reference interval, 2.6 to 5.0 g/dL). Six cats (4 with proven histoplasmosis) died or were euthanized, with survival times of 2 to 42 days after diagnosis (median, 16 days). The remaining 4 cats (2 with proven histoplasmosis) were still alive at the time of manuscript preparation 7 to 11 months after diagnosis (median, 10 months). Cats were treated with voriconazole (n = 4; 2 survivors), itraconazole (4; 1 survivor), and fluconazole (2; 1 survivor).

The 10 cats in the control group were considered to be true negatives. These cats ranged in age from 1 to 15 years (median, 11 years), and there were 5 neutered male and 5 female (4 neutered) cats. Definitive diagnoses for this population included small cell gastrointestinal lymphosarcoma (n = 3), lymphoplasmacytic enteritis (1), feline asthma (1), tularemia (1), and idiopathic hypercalcemia (1). One cat was diagnosed with nonassociative immune-mediated thrombocytopenia, with a platelet count of 2,000 platelets/µL (reference interval, 140,000 to 400,000 platelets/µL), and resolved with long-term prednisolone administration. No definitive diagnosis was obtained in the remaining 2 cats. One of these cats had fever and generalized lymphadenomegaly, which was cytologically interpreted as lymphoid hyperplasia. This cat had spontaneous resolution of disease with no therapy pending test results for histoplasmosis. The other cat had an acute onset of vomiting and diarrhea, which also spontaneously resolved with no therapy pending test results for histoplasmosis. This cat had been previously diagnosed and treated for histoplasmosis 1 year earlier and had a negative UA-EIA result 3 months earlier and was not deemed to be out of remission.

The RT-PCR on EDTA-collected whole blood was negative in all 20 cats. This included the sample in which organisms consistent with Histoplasma were seen in neutrophils and monocytes on a blood smear. The UA-EIA results were positive in all 10 cats with histoplasmosis, with a median value of 12.5 ng/mL (range, 0.35 to > 20 ng/mL; quantifiable reference interval, 0.2 to 20.0 ng/mL), and negative in all 10 control cats. The lowest UA-EIA result (0.35 ng/mL) was in a cat with cytological confirmation of histoplasmosis.

Serum IgG-EIA results were positive in 12 cats, including 9 cats with histoplasmosis and 3 control cats. The median IgG-EIA for cats with histoplasmosis was 25.1 enzyme immunoassay units (EU; range, 0 to > 80 EU; reference, < 8.0 EU is considered negative, ≥ 10.0 EU is considered positive). The median IgG-EIA for the control cats was 0.0 EU (range, 0.0 to 31.6 EU). Four control cats had a detectable IgG-EIA (range, 9.6 to 31.6 EU) and 3 were ≥ 10.0 EU. One control cat with an elevated IgG-EIA (31.6 EU) cohabitated with a cat that had been previously diagnosed and treated for histoplasmosis. This cat was diagnosed with idiopathic hypercalcemia and successfully managed with diet. Another control cat with an elevated IgG-EIA (22.5 EU) was in remission for previously diagnosed and treated histoplasmosis and had self-limiting acute-onset vomiting and diarrhea. The lowest IgG-EIA in this group (9.6 EU) was in a 1-year-old cat with tularemia. The fourth cat was diagnosed with small cell gastrointestinal lymphosarcoma (IgG-EIA, 21.4 EU).

The sensitivity for RT-PCR and sensitivity, specificity, positive and negative predictive values, and accuracy for IgG-EIA are listed in Table 1. There was a significant difference between the sensitivity of EIA to RT-PCR (P < .0001) and between IgG-EIA and RT-PCR (P = .0016). There was not a significant difference between the sensitivity of IgG-EIA and UA-EIA (P = .2208).

Discussion

In the population of cats for this study, the whole blood RT-PCR had 0% sensitivity for the diagnosis of feline histoplasmosis (all other statistical calculations for RT-PCR were considered not applicable). This was an unexpected finding given the performance of PCR for the detection of Histoplasma in peripheral whole blood in human patients, with sensitivities ranging as low as 18% and as high as 97% but with most studies demonstrating sensitivity around 60% to 70%.^{14–16,19,20} Possible reasons for the poor diagnostic sensitivity of the RT-PCR evaluated in the current study include inaccurate diagnoses, insufficient quantity of whole blood submitted for RT-PCR, ineffective Histoplasma DNA purification and isolation from whole blood, possible presence of PCR inhibitors in whole blood, and poor primer pair selection for RT-PCR.

A misdiagnosis in all 10 cats diagnosed with histoplasmosis seems an unlikely explanation for the poor performance of the RT-PCR. Although isolation and identification by culture were not performed, direct cytological visualization of organisms consistent with Histoplasma within macrophages was obtained in 60% (6/10 cats) of the overall presumed infected population. The 4 cats with a probable diagnosis of histoplasmosis, therefore, could have been misdiagnosed, although no alternative diagnosis was uncovered. Two of these cats had been treated long-term with prednisolone, 1 cat had a previous confirmed diagnosis of histoplasmosis, and the fourth cat had thoracic radiographic findings consistent with histoplasmosis. The UA-EIA was above the limit of quantification (> 20 ng/mL) in 2 of these 4 cats and 4.31 and 8.15 ng/mL in the other 2 cats. Two of these cats are still alive with resolution of clinical signs and a negative UA-EIA result after therapy with itraconazole (n = 1) and voriconazole (1). A necropsy was not performed on the other 2 cats with probable histoplasmosis that were euthanized 18 and 22 days after a positive UA-EIA result was obtained. However, it does not diminish the fact that the RT-PCR would still have a sensitivity of 0% if these 4 cats were excluded.

It is possible that insufficient sample size contributed to the poor sensitivity of the RT-PCR. The minimum sample submitted in this study was stated to be 1.5 mL, which is below the volume requested by the laboratory for RT-PCR (2 mL).¹⁷ The authors obtained the residual whole blood after performance of a routine CBC, mimicking common practice to add on the RT-PCR after the CBC and serum biochemistry failed to provide a diagnosis. The residual volume was estimated by visual inspection only, so it’s possible that the volume may have been between 1.0 and 1.5 mL. Volumes that appeared to be insufficient (obviously < 1.5 mL) on visual inspection prompted the collection of additional blood as per IACUC approval. The authors estimated that the residual volume would approximate 1.5 mL in a standard EDTA-anticoagulated blood tube that was filled to the recommended fill of 2.0 mL (assuming 250 µL for a single run on an automated analyzer, 150 µL for a microhematocrit tube, and 50 µL used to prepare a standard blood slide). Recognizing that the uncooperative nature of some cats results in insufficient blood volume obtained to fill an EDTA blood tube, the authors opted not to subject any cat to additional venipuncture if the residual volume was not obviously < 1.5 mL. The authors believe that this volume discrepancy was unlikely to contribute to the poor sensitivity of the RT-PCR in this study. A whole blood sample of 200 µL resulted in a sensitivity of 70% in a study¹⁴ comparing different nested-PCR protocols in humans with confirmed histoplasmosis, and a whole blood volume of 1.3 mL resulted in a sensitivity of 43.3% in a study¹⁶ evaluating the RT-PCR in human patients suspected of having histoplasmosis. We anticipated similar or better performance of the RT-PCR in cats as disseminated disease is common. Minimally, even with a sample volume of 0.5 mL, the authors would have expected a positive RT-PCR result in the 1 cat with abundant Histoplasma organisms present on a blood smear.

The most likely explanation for the poor performance of the RT-PCR is an ineffective DNA isolation and purification step. The authors suspect that in evaluation of protocols to use on EDTA-anticoagulated whole blood, blood samples from healthy cats were spiked with known concentrations of H capsulatum, as has been previously described.²¹ This method of test development may create confidence in test performance without evaluating real-world whole blood samples in which Histoplasma organisms are contained within monocytes and neutrophils and may be more difficult to isolate. Using whole blood from humans with histoplasmosis, 1 study¹⁴ suggested good recovery using proteinase K and a commercial kit for DNA extraction, and another study¹⁶ described superior DNA recovery using bead beating as compared to nonmechanical lysis. The authors are not aware of any studies using whole blood from cats with histoplasmosis describing the optimal technique to isolate and purify Histoplasma DNA.

Similarly, poor DNA purification techniques could result in the presence of PCR inhibitors in EDTA-anticoagulated whole blood specimens that may have resulted in false negative results. One study¹⁵ identified 7 EDTA-anticoagulated whole blood specimens from humans with histoplasmosis, 5 of which were culture-positive, that tested negative due to the presence of PCR inhibitors. Another study²² assessed the effects of human whole blood on RT-PCR and found that hemoglobin and IgG affect both amplification and fluorescence detection. The authors believe this to be an unlikely explanation, however, as EDTA-anticoagulated whole blood does not appear to affect recovery from spiked healthy control samples. The presence of anti–Histoplasma IgG seems an unlikely inhibitor as the RT-PCR was still negative in the 1 cat with a negative IgG-EIA.

Finally, the authors believe poor primer pair selection or the presence of unique geographic clades that are not amplified by the standard primer pairs that identify H capsulatum is an unlikely explanation for the failure of the RT-PCR in this study. Although the primer pairs used for the RT-PCR evaluated in this study are not published, the authors are confident that appropriate primer pairs were used by the commercial laboratory and would have been successful in amplification during initial test evaluation. Likewise, the authors are not aware of evidence of unique clades failing to be amplified by commonly used primer pairs.⁵

The IgG-EIA has historically been shown to have good sensitivity (81.1%) and specificity (94.7%) for the diagnosis of histoplasmosis in cats.¹³ In the current study, however, the IgG-EIA yielded slightly higher sensitivity (90%) and lower specificity (70%). This result likely reflects a difference in the population characteristics, which included previously infected cats in both groups in the current study. Larger studies that include more healthy cats may identify a lower specificity for the diagnosis of histoplasmosis given that exposure in certain regions can be high. Regardless, serology should only be used as a supportive diagnostic tool and not as a sole agent for the diagnosis of histoplasmosis in cats.

A limitation of this study was the small sample size. The initial goal was to enroll 20 cats with histoplasmosis on the assumption that RT-PCR would have 70% sensitivity. The study was terminated early when it was determined that enrolling an additional 10 cats would not alter the results of the study. With the results reported above, a larger study assessing this particular RT-PCR assay would have questionable utility.

The authors’ inability to access the protocol used for the RT-PCR to understand the reasons why this test did not perform was a clear limitation. Samples from liver, spleen, and peripheral lymph nodes were not concurrently submitted for RT-PCR; however, these results could have provided more insight as to whether the DNA isolation/purification procedure or PCR primer pairs were responsible for the poor performance of the whole blood RT-PCR.

In conclusion, the whole blood RT-PCR should not be used for the diagnosis of feline histoplasmosis. Further research and development would be required to formulate a sensitive whole blood RT-PCR assay. In addition, a positive IgG-EIA should not be considered diagnostic for histoplasmosis in cats.

Disclosures

Dr. Hanzlicek currently serves as the Director of Veterinary Medicine and Research at MiraVista Diagnostics, which commercially offers the Histoplasma Antigen EIA and Histoplasma Feline Antibody IgG-EIA described in this manuscript. The other authors have nothing to disclose.

No AI-assisted technologies were used in the generation of this manuscript.

Funding

Funding for Histoplasma Antigen EIA and Histoplasma Feline Antibody IgG-EIA tests were provided by MiraVista Diagnostics. Funding for the remainder of the study was provided from internal sources.

ORCID

K. Harkin https://orcid.org/0000-0003-4843-357X