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Preliminary evaluation of a novel enzyme immunoassay for the detection of Histoplasma capsulatum antigen in urine.

Ryan J. Welch1, Elitza S. Theel2, Marc Roger Couturier1,3
1Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology, Salt Lake City, UT
2Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN
3Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT

histoplasmosis test

What is Histoplasmosis

Histoplasmosis is caused by Histoplasma capsulatum, a fungus endemic to the Mississippi and Ohio River valleys as well as the southern United States and parts of Central and South America. With the seropositivity rates in endemic regions being high and immunocompromised individuals at the greatest risk of morbidity and mortality, serology has limited diagnostic utility. Therefore, urine antigen detection assays have become adjunct methods of detection that are particularly useful in disseminated disease.

Recently, Niche Diagnostics (Scarborough, ME) has developed an antigen detection enzyme immunoassay (EIA) using a preparation of two monoclonal antibodies targeted against a specific H. capsulatum glycoprotein, which includes galactommanan. In this study we evaluate these reagents against previously established Histoplasma antigen detection assays.


METHODS

Urine samples (n=224) were collected at Mayo Clinic and submitted for H. capsulatum testing to MiraVista Diagnostics (MVista Histoplasma Ag quantitative EIA, Indianapolis, IN) as well as performed at Mayo Medical Laboratories on the Histoplasma Galactomannan Antigen EIA assay (IMMY HGM, IMMY Diagnostics, Norman, OK). Each sample was split and sent to ARUP Laboratories to be tested using the Niche reagents. All specimens were stored at -80°C prior to testing and deidentified and handled as per our Mayo Clinic and ARUP Laboratories approved IRBs.

MVista Histoplasma Ag quantitative EIA

All specimens were tested on MVista Histoplasma Ag quantitative EIA. Results are quantitatively reported in ng/mL between 0.4 ng/mL and 19 ng/mL (detected results outside the analytical range are reported but not quantified).

IMMY HGM assay

All specimens were tested on the IMMY HGM assay according to the manufacturer’s instructions for use. Briefly, undiluted urine is added to microtiter wells coated with monoclonal antibody specific to galactomannan and incubated for one hour at 37°C. Following a wash step, horseradish peroxidase (HRP) conjugated anti-galactomannan antibody is added and incubated. Following a second wash step,3,3′,5,5′-tetramethylbenzidine (TMB) is added, incubated for 30 minutes at room temperature. Stop solution (2 N sulfuric acid) is added and the wells are analyzed spectrophotometrically at 450 nm. Calibrators provided by the manufacturer allow quantitation in ng/mL. Based on previously published experiments, specimens that quantitate between 0.11 ng/mL and 0.49 ng/mL are considered indeterminate, results below this range are negative, and above are positive.

Niche Histoplasmosis Urinary Antigen ELISA

All specimens were tested on the Niche Histoplasma assay. Briefly, undiluted urine is added to microtiter wells containing two unique monoclonal antibodies specific to H. capsulatum glycoprotein. The wells are washed after a one hour incubation with shaking at room temperature. A solution containing biotinylated versions of the same antibodies bound to each well is then added and incubated for one hour, while being shaken at room temperature. Following a second wash step, HRP conjugated streptavidin is then added and again incubated at room temperature for one hour. Following a final wash step, TMB substrate is added, incubated for 10 minutes and stopped with HCl. The wells are then analyzed spectrophotometrically at 450 nm. Calibrators provided by the manufacturer allow for quantitation in ng/mL from 0.5 ng/mL to 65.0 ng/mL. Results below the limit of quantification are considered negative.


RESULTS

When compared to MVista results, the Niche assay had an overall agreement of 86% on all 224 specimens (Table 1).

Samples that are indeterminate on the IMMY HGM assay have shown variable agreement compared with MVista in past studies. A total of 150 IMMY indeterminate samples were tested on both the Niche and MVista assays.

In samples indeterminate on the IMMY HGM assay but tested positive on the Niche assay, 44 of 59 (75%) agreed as positive with MVista. In samples indeterminate on the IMMY HGM assay that were negative on the Niche assay 86 of 91 (95%) agreed as negative on the MVista assay (Table 2).

On all other specimens (not indeterminate on the IMMY HGM assay) the Niche assay compared to MVista with an overall agreement of 85% (63 of 74), while the IMMY HGM assay compared to MVista with an overall agreement of 89% (66 of 74).

Both the IMMY HGM assay and the Niche assay had low agreement on specimens that were not indeterminate by the IMMY HGM assay (Table 3).

CONCLUSIONS

Based on the data presented, Niche Diagostics has developed reagents that show potential in detecting Histoplasma capsulatum antigen in urine specimens.

In problematic samples that were indeterminate on the IMMY HGM assay, the Niche assay may provide added benefit in that it was able to generate results for 130 of 150 specimens that were in agreement with MVista, but would not have had a conclusive result if only the IMMY HGM assay was used. The majority of specimens that did not agree with MVista in were positive values (15 of 20). These could represent false-positive values, or true positives that are a result of different assay target specificity. Additional studies will be required to clearly determine the significance of these values.

In samples that did not fall in the indeterminate range on the IMMY HGM assay, both the IMMY HGM assay and the Niche assay revealed false negatives when compared with the MVista assay.

Overall the Niche assay may provide added benefit over the IMMY HGM assay, particularly in low positive specimens (specimens indeterminate on the IMMY HGM assay). However, further refinement of the assay to eliminate any possible false negatives is warranted. Testing serial monitoring specimens from patients with Histoplasmosis is also required to test the ability of the Niche assay to detect trailing concentrations of antigen in appropriately treated patients.

ACKNOWLEDGMENTS

Evaluation reagents were graciously provided at no cost by Niche Diagnostics.

Works Cited
1. Theel ES, Harring JA, Dababneh AS, Rollins LO, Bestrom JE, Jespersen DJ. 2015. Reevaluation of commercial reagents for detection of Histoplasma capsulatum antigen inurine.

2. Kauffman CA. 2007. Histoplasmosis: a clinical and laboratory update. Clinical microbiology reviews 20:115-132.

3. Theel ES, Jespersen DJ, Harring J, Mandrekar J, Binnicker MJ. 2013. Evaluation of an enzyme immunoassay for detection of Histoplasma capsulatum antigen from urine specimens. Journal of clinical microbiology 51:3555-3559.

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We will look for opportunities to develop technology for emerging disease that because of the limited initial market testing is limited or nonexistent.

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Table 1: Agreement between MVista and the Nicheassay on all study specimens.

 
MVista
NichePositiveNegativeTotal
Positive581674
Negative15135150
Total73151224

Table 2: Agreement between MVista and the Nicheassay on samples that were indeterminate by the IMMY HGM assay.

 
MVista
NichePositiveNegativeTotal
Positive441559
Negative58691
Total49101150

Table 3: Agreement between Niche, IMMY HGM and MVista on samples not indeterminate by the IMMY HGM assay.

 
MVista
NichePositiveNegativeTotal
Positive14115
Negative104959
Total245074
IMMY HGM
Positive14014
Negative85260
Total225274
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