Pretreatment Histoplasma capsulatum urine enzymatic immunoassay concentrations do not correlate with outcome but may be influenced by renal function in cats with histoplasmosis

Abstract

Objectives

The objective of this study was to determine if urine Histoplasma antigen (HAg) enzyme immunoassay (EIA) concentrations at the time of diagnosis and prior to the administration of antifungal agents are predictive of outcome for cats infected with Histoplasma capsulatum and to determine if compromised renal function affects urine HAg EIA measurements.

Methods

Medical records at four institutions were searched to identify cats diagnosed with histoplasmosis between April 2012 and December 2015. Pretreatment urine Histoplasma EIA values were recorded, along with patient signalment, serum creatinine concentration, urine specific gravity, site(s) of infection and survival data.

Results

Pretreatment urine HAg EIA measurements were available for 50 cats, and ranged from 0–19.1 ng/ml (median 6.3 ng/ml). Thirty-five cats were alive at day 180, 12 had died or were euthanized (median survival time 24 days; range 2–124 days) and three were lost to follow-up. The median urine HAg EIA at the time of diagnosis for cats alive at 6 months was 5 ng/ml (range 0–19.1); this was similar to findings for the non-survivors (median 7.29 ng/ml; range 0.78–19.1; P = 0.54). Surviving cats were significantly younger (mean age 6.9 years) than non-survivors (mean age 9.9 years; P = 0.03) but median body weights (3.8 kg vs 3.6 kg) and rates of pulmonary involvement (22/35 vs 9/12) were similar for the two groups. Median urine HAg EIA concentration was lower in cats with evidence of renal compromise than cats with acceptable renal function (0.54 ng/ml vs 7.2 ng/ml; P <0.013).

Conclusions and relevance

Urine HAg EIA concentrations at the time of diagnosis are not predictive of outcome in cats with histoplasmosis and should not be used as a prognostic indicator in this species. Renal function may influence urine HAg EIA concentrations in cats; further investigation is needed to see if concurrent kidney disease impacts test sensitivity.

Introduction

Histoplasma capsulatum is a dimorphic soil-borne fungus, and is widely distributed in temperate and subtropical regions. Infection with this organism may be subclinical and transient or cause a range of signs, reflecting both location and severity. Common sites of infection in cats include the lungs, spleen, gastrointestinal tract, eye and skin.^1–3 Traditionally, a diagnosis was established by the direct visualization of organisms in affected tissues or within circulating monocytes. However, in the last decade, non-invasive tests based on the detection of antigens in serum, urine and other body fluids have become available, ⁴ and a Histoplasma antigen (HAg) enzyme immunoassay (EIA) is routinely used to document infection and to monitor the response to treatment in cats with disseminated histoplasmosis. ⁵ Any level of antigenuria/antigenemia is considered indicative of active disease, and the urine HAg EIA has a >90% sensitivity for non-ocular infection in this species.^2,6 In addition, antigenuria significantly decreases with effective treatment and can be used to indicate disease remission in cats. ⁵

Successful treatment of histoplasmosis requires prolonged administration of antifungal agents, and requires regular follow-up visits and laboratory testing.^1,7 Many cats require substantial supportive care, including feeding tube placement, and costs can be considerable. Although a recent study indicated that severe respiratory, hepatic, hematologic or neurologic impairment was associated with a negative prognosis, ⁸ an objective prognostic indicator, readily available at the time of diagnosis, would be helpful for both owners and veterinarians and could guide appropriate decision-making for affected cats. Negative prognostic factors in human patients with disseminated histoplasmosis include hypotension, thrombocytopenia, hyperlactatemia, dyspnea and renal insufficiency.^9,10 In addition, the level of Histoplasma antigenuria/antigenemia appears to correlate with the severity of infection in people. ¹¹

The primary objective of this study was to determine if urine EIA concentrations at the time of diagnosis are predictive of outcome (ie, survival to 6 months) for cats treated for histoplasmosis. A secondary objective was to determine the effect of renal compromise on levels of antigenuria in this species.

Materials and methods

The electronic medical records databases at four veterinary hospitals were searched to identify cats with a diagnosis of histoplasmosis and a contemporaneous, pretreatment urine HAg EIA result. All urine EIA testing was performed at a single laboratory (MiraVista Diagnostics, Indianapolis, IN, USA) using the same, quantitative sandwich methodology. ¹² Information regarding patient age, sex and weight was recorded, along with the affected organ(s). The contemporaneous serum creatinine concentration and urine specific gravity (USG) were also recorded, when available. The medical record was reviewed for evidence of fluid therapy (intravenous or subcutaneous) in the 24 h prior to measurement of USG. Cats were classified as having kidney disease if a pre-fluid USG was <1.035 and serum creatinine concentration was ⩾140 µmol/l (1.6 mg/dl); cats were classified as having acceptable renal function if USG was ⩾1.035 and serum creatinine concentration was <140 µmol/l (<1.6 mg/dl). ¹³ Antifungal treatments were listed. Cases were excluded if the cat received systemic antifungal medications for any reason in the previous 6 months.

Cats were classified as survivors if they were alive 180 days after the date of collection of the urine sample used for HAg EIA measurement. Cats were classified as non-survivors if they died or were euthanized after beginning appropriate treatment for histoplasmosis, with survival time counted as days from submission of urine for testing and date of death.

For statistical purposes, positive urine HAg EIA results above the level of quantification (19 ng/ml) were listed as 19.1 ng/ml and those below the level of quantification (0.4 ng/ml) were listed at 0.3 ng/ml. A commercially available software program (GraphPad Prism) was used for statistical analysis, with significance set at P <0.05. Data sets were tested for normality using the Pearson test. Parametric data are expressed as mean ± SD. Non-parametric data are expressed as median and range, and were compared using the Mann–Whitney U-test.

Results

A total of 50 cats met the inclusion criteria. Ages ranged from 1–18 years, with a median of 7 years. Twenty-six cats (52%) were female (25 spayed; one intact) and 24 (48%) were castrated males. Body weights ranged from 2–7.8 kg (median 3.7 kg). Thirty-one cats (62%) had evidence (based on imaging findings or clinical signs) of pulmonary involvement, 11 cats (22%) had cytologically confirmed splenic or hepatic infection and 10 (20%) had findings suggestive of ocular involvement. All cats received either fluconazole (n = 33; 66%) or itraconazole (n = 17; 34%), often in combination with amphotericin B (n = 13; 26%).

Thirty-five cats (70%) were alive at day 180; 12 cats (24%) had died (n = 1) or had been euthanized (n = 11) before day 180 (median survival time 24 days; range 2–124 days), and three cats were lost to follow-up. Survival rates (ie, percentage of cats known to be alive at day 180) for the four hospitals ranged from 57–86%. The median urine HAg EIA at the time of diagnosis for the survivors was 5 ng/ml (range 0–19.1 ng/ml); this was similar to that for non-survivors (median 7.29 ng/ml; range 0.78–19.1; P = 0.54). Six of the 35 survivors (17%) had a urine Histoplasma EIA above the assay limit of 19 ng/ml. Additionally, survival rates for cats with urine HAg EIA above (n = 22) or below (n = 25) 10 ng/ml were similar (77% vs 73%). Surviving cats were significantly younger (mean age 6.9 years; range 1–14 years) than non-survivors (mean age 9.9 years; range 4–18 years; P = 0.03), but median body weights (3.8 kg vs 3.6 kg) and rates of pulmonary involvement (22/35 [63%] vs 9/12 [75%]) were similar.

Serum creatinine concentrations were measured at the time of collection of urine for HAg EIA testing in 42 cats (84%) and ranged from 44–205 µmol/l (0.5–2.3 mg/dl), with a median of 97 µmol/l (1.1 mg/dl). A pre-fluid USG was listed for 17 cats (34%). On the basis of USG and serum creatinine concentration, four of these cats (23%) had evidence of renal compromise (ie, pre-fluid USG <1.035 and serum creatinine ⩾140 µmol/l [⩾1.6 mg/dl]) and 13 (77%) had apparently acceptable renal function (ie, USG ⩾1.035 and serum creatinine <140 µmol/l [<1.6 mg/dl]). Serum creatinine concentrations in the cats with evidence of renal compromise ranged from 141–203 µmol/l (1.6–2.3 mg/dl). None of these cats had received diuretic agents or drugs expected to impact renal concentrating ability. Median urine HAg EIA concentration for the cats with evidence of renal compromise was 0.54 ng/ml (range 0–0.97 ng/ml); this was significantly (P <0.013) lower than that for the cats with acceptable renal function (median 7.2 ng/ml; range 0.3–19.1 ng/ml).

Discussion

Overall survival rates for the cats in this study were encouraging, with 35/50 (70%) known to be alive 6 months post-diagnosis. In this study population, pretreatment Histoplasma antigenuria levels were not predictive of outcome, with similar median urine HAg EIA values for survivors and non-survivors. Although the number of cats reported here is relatively small and the study may have been underpowered, several cats (17%) in the survivor group had urine EIA concentrations beyond the upper limit of the assay (ie, >19 ng/ml), suggesting that this value should not discourage an owner from treating an affected cat.

The results of this study suggest that urine HAg EIA concentrations may be affected by underlying renal function, as the median measurement for cats with evidence of kidney disease was significantly lower than for cats with apparently normal renal status. This may reflect the impact of USG on antigen concentrations, differences in renal blood flow and antigen delivery to the kidneys, or in renal handling of filtered fungal antigens. However, the number of cats with sufficient data to draw conclusions about renal status was small, and our findings in this regard should be interpreted with caution. It is also difficult to assess renal function on the basis of a single time point, and without any information on patient hydration status, and so on.

A major limitation of this study is its retrospective nature. Patients were not subjected to a standardized diagnostic evaluation, pretreatment data points were not available for every case, and decisions regarding treatment were made by the attending clinician. Also, the reason for euthanasia in the non-survivor group was not routinely recorded. It is assumed that this decision reflected the condition of the cat, but it may instead have been driven by financial concerns or comorbid conditions. It is interesting to note that older cats were less likely to be alive at 180 days; this may reflect owner reluctance to treat or the presence of concurrent disease(s).

Prospective studies are needed to identify prognostic parameters for cats with histoplasmosis and to investigate the impact of renal function on the sensitivity of antigenuria-based diagnostic tests in this species.

Conclusions

This study suggests that pretreatment urine HAg EIA concentrations are not predictive of outcome in cats treated for histoplasmosis.