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Clinical and Vaccine Immunology, February 2008, p. 392-393, Vol. 15, No. 2
1071-412X/08/$08.00+0     doi:10.1128/CVI.00383-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cryptococcal Glucoxylomannan Does Not Exhibit Cross-Reactivity in the MVista Histoplasma Antigen Enzyme Immunoassay{triangledown}

David Zhuang,1 Chadi Hage,2 Magdia De Jesus,3 Emily Hackett,1 Michelle Durkin,1 Thomas E. Davis,4 Arturo Casadevall,3 and L. Joseph Wheat1*

MiraVista Diagnostics, Indianapolis, Indiana 46241,1 Indiana University School of Medicine, Department of Pulmonary Medicine, Roudebush Veterans' Administration Hospital, Indianapolis, Indiana 46202,2 Albert Einstein College of Medicine, Bronx, New York 10461,3 Indiana University School of Medicine, Department of Pathology, Wishard Memorial Hospital, Indianapolis, Indiana 462024

Received 24 September 2007/ Returned for modification 10 November 2007/ Accepted 29 November 2007


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ABSTRACT
 
The potential for cross-reaction between Cryptococcus neoformans and Histoplasma capsulatum in antigen assays was evaluated. We tested patient samples, spleens from infected mice, and purified polysaccharides in the MVista Histoplasma antigen enzyme immunoassay and cryptococcal antigen latex agglutination system for cross-reactivity, and none was observed.


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TEXT
 
The diagnosis of invasive or systemic mycosis is often accomplished through the detection of polysaccharide antigens in body fluids by immunologic assays. The specific polysaccharides detected are galactomannans (GalM) of Aspergillus spp. (2) and Histoplasma capsulatum (1) and glucuronoxylomannan (GXM) of Cryptococcus neoformans (2). We have observed several cases in which tests for both cryptococcal GXM and Histoplasma GalM were positive and where dual infections were documented. However, the literature on dual infection is not extensive (3). This prompted us to investigate further the presence of cross-reactivity between cryptococcal GXM and Histoplasma GalM.

Cryptococcal antigen latex agglutination system (CALAS) kits were purchased from Meridian Bioscience (Cincinnati, OH) and used according to the manufacturer's instructions. The serum samples were treated with pronase before being tested with a CALAS kit. The MVista Histoplasma antigen enzyme immunoassay (EIA) was performed at MiraVista Diagnostics (Indianapolis, IN) as previously reported (1).

Histoplasma and Cryptococcus polysaccharide antigens were prepared and purified as previously described (1, 2). Histoplasma GalM and cryptococcal GXM, galactoxylomannan, and capsular polysaccharide were tested at a concentration of 1 µg/ml, and the CALAS kit positive control was tested undiluted. Cryptococcal GXM, galactoxylomannan, and capsular polysaccharide and the CALAS control were strongly positive (agglutination score of 3+ or 4+) by CALAS assay but negative in the Histoplasma antigen EIA. Conversely, Histoplasma GalM was positive in the Histoplasma antigen EIA (>39 ng/ml) but negative by CALAS assay.

Twenty-nine residual serum specimens from 15 patients with progressive disseminated histoplasmosis were tested by CALAS assay, in a study approved by the institutional review board of Clarian Hospital, Indianapolis, IN. All had underlying immunosuppression, including AIDS in 10, organ transplantation in 2, and miscellaneous causes in 3 patients. The basis for diagnosis of histoplasmosis was Histoplasma antigenemia and antigenuria in seven cases, culture in six cases, and histopathology in two cases. Histoplasma antigenemia ranged from <0.6 to >39 ng/ml (median, 2.5 ng/ml). No samples were positive by CALAS assay (Table 1).


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  		TABLE 1. Cross-reactivity in clinical specimens and experimental infection

Residual serum or cerebrospinal fluid samples from 25 patients with cryptococcosis were previously described (2). The CALAS assay was positive for 24 of 25 patients, at titers of 1:1 to 1:65,536 (median, 1:1,024). Culture was the basis for diagnosis for the 25th patient. None of the samples were positive in the Histoplasma antigen EIA (Table 1).

In a previously described murine histoplasmosis model (4), eight B6C3F1 mice were infected intranasally with 1 x 106 H. capsulatum organisms and euthanized on day 10 of infection according to institutional guidelines (4). Spleen tissue was homogenized in 2 ml of RPMI medium and tested in the Histoplasma antigen EIA following 10-fold dilution but used undiluted in the CALAS assay. Spleen tissues contained high levels of Histoplasma antigen but were negative by CALAS assay (Table 1).

In a cryptococcal experimental infection model (2), 12 BALB/c mice were infected intravenously with 1 x 104 C. neoformans strain 24067 organisms and then euthanized on day 7 of infection. The spleen tissue was homogenized in 2 ml of RPMI medium and tested undiluted in the Histoplasma antigen EIA and the CALAS assay. Spleen tissues were positive by CALAS assay but negative in the Histoplasma antigen EIA.

In conclusion, no cross-reactivity between Histoplasma GalM and cryptococcal polysaccharides was observed. When both the CALAS assay and MVista Histoplasma antigen EIA are positive, dual infection should be suspected. Note that these findings should not be extrapolated to other Histoplasma or cryptococcal antigen assays.


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ACKNOWLEDGMENTS
 
This work was supported by MiraVista Diagnostics, Indianapolis, IN, and by NIH grants AI33774, AI33142, and HL59842-01 to A.C. M.D. was supported by NCI/NIH training grant 2T32CA009173-31.

We disclose that L.J.W., D.Z., and E.H. are employees of MiraVista Diagnostics, a laboratory that performs the MVista Histoplasma antigen EIA.


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FOOTNOTES
 
* Corresponding author. Mailing address: MiraVista Diagnostics, 4444 Decatur Blvd., Suite 300, Indianapolis, IN 46241. Phone: (317) 856-2681. Fax: (317) 856-3685. E-mail: jwheat{at}miravistalabs.com Back

{triangledown} Published ahead of print on 12 December 2007. Back


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REFERENCES
 
    1
  1. Connolly, P. A., M. M. Durkin, A. M. LeMonte, E. J. Hackett, and L. J. Wheat. 2007. Improvement in the detection of Histoplasma antigen by a quantitative enzyme immunoassay. Clin. Vaccine Immunol. 14:1587-1591.[Abstract/Free Full Text]
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  2. De Jesus, M., E. Hackett, M. Durkin, P. Connolly, A. Casadevall, R. Petraitiene, T. J. Walsh, and L. J. Wheat. 2007. Galactoxylomannan does not exhibit cross-reactivity in the Platelia Aspergillus enzyme immunoassay. Clin. Vaccine Immunol. 14:624-627.[Abstract/Free Full Text]
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  3. Swaminathan, S., K. Imrit, J. Green, and K. Das. 2006. Concomitant disseminated histoplasmosis and cryptococcosis in a person with AIDS. AIDS Read. 16:602-606.[Medline]
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  4. Wheat, L. J., E. Hackett, M. Durkin, P. Connolly, R. Petraitiene, T. J. Walsh, K. Knox, and C. Hage. 2007. Histoplasmosis-associated cross-reactivity in the BioRad Platelia Aspergillus enzyme immunoassay. Clin. Vaccine Immunol. 14:638-640.[Abstract/Free Full Text]

Clinical and Vaccine Immunology, February 2008, p. 392-393, Vol. 15, No. 2
1071-412X/08/$08.00+0     doi:10.1128/CVI.00383-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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