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Clinical and Vaccine Immunology, October 2007, p. 1387-1388, Vol. 14, No. 10
1071-412X/07/$08.00+0     doi:10.1128/CVI.00267-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

LETTERS TO THE EDITOR

Evaluation of Reagents for Detection of Histoplasma capsulatum Antigenuria

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Lindsley and colleagues (5) reported an antigen assay for diagnosis of histoplasmosis. Although they do not claim to have compared their test to that at MiraVista Diagnostics (9), the reader may draw that conclusion since the authors note that I provided antibodies, vaccine strain, and patient specimens for their study (5). They reported superior results using their antibodies.

They did not compare their assay to the MiraVista Diagnostics assay, however. The antibodies that I provided them in 1991 were from rabbits used in our original assay (2, 8). The urine specimens were from patients with AIDS and progressive disseminated histoplasmosis (PDH) collected in 1983 to 1989, some of which were concentrated. Our original assay and those antibodies are now obsolete. In 2003, we discovered false-positive results caused by antirabbit antibodies (7). Subsequently we developed a second-generation assay that reduced false positivity by 75% (9).

The authors imply that false-positive results caused by antirabbit antibodies would not be relevant in urine. False Legionella antigenuria was reported in a transplant patient treated with anti-thymocyte globulin (1), and we have observed false Histoplasma antigenuria in a myeloma patient. In that case, the urine was highly positive in our original assay but negative in our second-generation assay. That urine also contained antirabbit antibodies.

False antigenemia or antigenuria is not a trivial problem. Kricka concluded "Efforts should be directed at improving methods for identifying and eliminating this type of analytical interference (3)" and that "manufacturers of immunoassay test kits...must be agreed to...refinements of immunoassays to render them interference free (4)." Use of an inferior test may lead to preventable testing errors.

What's less appreciated is the enhanced sensitivity of our second-generation assay. Improved sensitivity is desirable because false-negative results occurred in 20% of non-AIDS patients with PDH and two-thirds with pulmonary histoplasmosis (2, 10). We have produced antibodies that detect purified Histoplasma galactomannan with two- to fourfold-higher sensitivity than our original antibodies. Sensitivity in patients also is superior. Among patients with AIDS and PDH (6), antigenuria was detected in 100% of patients in our second-generation assay versus 94% in our original assay and antigenemia was detected in 95% versus 87% (P = 0.014), respectively (Fig. 1). Furthermore, 15 of 18 (83%) serum specimens and 24 of 33 (73%) urine specimens, including baseline specimens or ones obtained during treatment, that were negative in the original assay were positive in the second-generation assay.

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FIG. 1. Histoplasma antigen in the original versus the second-generation MiraVista assay in AIDS patients with PDH. The left panel shows antigen in the baseline serum (n = 36) and urine (n = 38) specimens. Results for individual cases are connected by the diagonal lines, and points above the horizontal line at 1 unit (EU) are positive. The median antigen level in serum was 5.7 EU (95% confidence interval [CI], 2.4 to 11.6 EU) in the original versus 39.7 EU (95% CI, 19.9 to 50.5 EU) in the second-generation assay, and in urine, the level was 11.7 EU (95% CI, 9.2 to 12.9 EU) in the original versus 51.4 EU (95% CI, 42.5 to 58.5 EU) in the second-generation assay. The right panel shows antigen in serum (n = 18) and urine (n = 33) specimens obtained at enrollment or during therapy that were negative in the original assay (vertical axis from 0.1 to 10 EU because all results are <10 EU).

Comparison of their findings (5) to ours (Fig. 1) shows the two assays differ greatly. For example, their positive results in AIDS patients with PDH ranged from 1.4 to about 3.5 times their normal control. Positive results are always at least twice and often (30%) >100 times the normal control in our assay. Furthermore, specificity was not assessed in their assay but is about 98% in our assay, excluding other endemic mycoses. In summary, the recently reported Histoplasma antigen assay (5) has not been compared to the MiraVista assay (9) but appears to be less sensitive.

 
L. Joseph Wheat is the president of MiraBella Technologies, the developer of the second generation Histoplasma antigen assays, and MiraVista Diagnostics, a laboratory that does fungal antigen testing.

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1. Deforges, L., P. Legrand, J. Tankovic, C. Brun-Buisson, P. Lang, and C. J. Soussy. 1999. Case of false-positive results of the urinary antigen test for Legionella pneumophila. Clin. Infect. Dis. 29:953-954.[Medline]
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2. Durkin, M. M., P. A. Connolly, and L. J. Wheat. 1997. Comparison of radioimmunoassay and enzyme-linked immunoassay methods for detection of Histoplasma capsulatum var. capsulatum antigen. J. Clin. Microbiol. 35:2252-2255.[Abstract]
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3. Kricka, L. J. 1999. Human anti-animal antibody interferences in immunological assays. Clin. Chem. 45:942-956.[Abstract/Free Full Text]
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4. Kricka, L. J. 2000. Interferences in immunoassay—still a threat. Clin. Chem. 46:1037-1038.[Free Full Text]
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5. Lindsley, M. D., H. L. Holland, S. L. Bragg, S. F. Hurst, K. A. Wannemuehler, and C. J. Morrison. 2007. Production and evaluation of reagents for detection of Histoplasma capsulatum antigenuria by enzyme immunoassay. Clin. Vaccine Immunol. 14:700-709.[Abstract/Free Full Text]
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6. Wheat, J., S. MaWhinney, R. Hafner, D. McKinsey, D. Chen, A. Korzun, K. J. Shakan, P. Johnson, R. Hamill, D. Bamberger, P. Pappas, J. Stansell, S. Koletar, K. Squires, R. A. Larsen, T. Cheung, N. Hyslop, K. K. Lai, D. Schneider, C. Kauffman, M. Saag, W. Dismukes, W. Powderly, et al. 1997. Treatment of histoplasmosis with fluconazole in patients with acquired immunodeficiency syndrome. Am. J. Med. 103:223-232.[CrossRef][Medline]
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7. Wheat, L. J., P. Connolly, M. Durkin, B. K. Book, A. J. Tector, J. Fridell, and M. D. Pescovitz. 2004. False-positive Histoplasma antigenemia caused by antithymocyte globulin antibodies. Transplant. Infect. Dis. 6:23-27.[CrossRef][Medline]
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8. Wheat, L. J., R. B. Kohler, and R. P. Tewari. 1986. Diagnosis of disseminated histoplasmosis by detection of Histoplasma capsulatum antigen in serum and urine specimens. N. Engl. J. Med. 314:83-88.[Abstract]
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9. Wheat, L. J., J. Witt III, M. Durkin, and P. Connolly. 2007. Reduction in false antigenemia in the second generation Histoplasma antigen assay. Med. Mycol. 45:169-171.[CrossRef][Medline]
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10. Williams, B., M. Fojtasek, P. Connolly-Stringfield, and J. Wheat. 1994. Diagnosis of histoplasmosis by antigen detection during an outbreak in Indianapolis, Ind. Arch. Pathol. Lab. Med. 118:1205-1208.[Medline]

						L. Joseph Wheat MiraVista Diagnostics 4444 Decatur Blvd. Indianapolis, Indiana 46241
						Phone: (317) 856-2641, ext. 452, E-mail: jwheat{at}miravistalabs.com

Authors’ Reply

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We thank Dr. Wheat for his attention to our publication. We appreciate his recognition that we did not compare our assay to the current MiraVista assay. At this time, we are reluctant to discuss the relative sensitivities of these assays without obtaining additional supporting data. At present, we are in the process of conducting a clinical evaluation of our assay and will be pleased to share the results in an appropriate future publication. We thank Dr. Wheat for his comments and for his many contributions to the field of fungal diagnostics. We all agree that it is most important to produce reliable reagents that can be used to diagnose disseminated histoplasmosis, particularly in human immunodeficiency virus-infected individuals. These patients are ever more frequently found in developing countries where individuals have the least access to quality care and affordable testing. The availability of diagnostic reagents in these countries would be an important step in decreasing morbidity and mortality from histoplasmosis in these regions.

						Mark D. Lindsley* Heather L. Holland Sandra L. Bragg Steven F. Hurst Kathleen A. Wannemuehler Christine J. Morrison Division of Foodborne, Bacterial, and Mycotic Diseases Centers for Disease Control and Prevention 1600 Clifton Rd. Mailstop G11 Atlanta, Georgia 30333
						* Phone: (404) 639-4340, Fax: (404) 639-3546, E-mail: MLindsley{at}cdc.gov

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Clinical and Vaccine Immunology, October 2007, p. 1387-1388, Vol. 14, No. 10
1071-412X/07/$08.00+0     doi:10.1128/CVI.00267-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wheat, L. J. Articles by Morrison, C. J. Search for Related Content PubMed PubMed Citation Articles by Wheat, L. J. Articles by Morrison, C. J.