Abstract

Dear Editor, We are writing about the recently published article "Antibodiesagainst Stachybotrys chartarum extract and its antigenic components, Stachyhemolysin and Stachyrase-A:a new clinical biomarker" [1]. The author states that his hypothesis is "that environmental exposureto Stachyhemolysin and Stachyrase-A may result in antibody production against these mold antigens." Thereare numerous methodologic deficiencies in this paper which preclude drawing any conclusions regardingthe utility of anti-stachyhemolsyin and anti-stachyrase-A antibodies as biomarkers. First, and most basically,it is unknown if the sera that were tested for these antibodies came from individuals with documentedexposure to Stachybotrys chartarum, i.e. persons who had spent considerable time in an environment withsignificant Stachybotrys chartarum contamination. Because there are no acceptable biomarkers of exposureto Stachybotrys chartarum in humans [2], it is impossible to know if the patients had any Stachybotryschartarum exposure. The sera used are all from persons with very high IgE levels and positive for IgEto different mold extracts, although which mold extracts are not reported. These individuals would notbe representative of the general population who would be exposed toStachybotrys chartarum. The authorsuggests that the observed antibody reactivity to their antigen preparations make them possible clinicalmarkers without showing the reactivity of other mold-sensitive but Stachybotrys -negative sera or showingany data on the species-specificity of the antibodies they detect. There is enormous cross-reactivityamong IgG and IgM from related and even unrelated fungal species [3], making it impossible to determinewhether their primary sensitization was toStachybotrys chartarum. Second, there are no controls, or unexposedsera tested as comparison. It appears that the author is using the presence of IgG to Stachybotrys chartarumas his "gold standard" for exposure; however, this has been shown not to be a useful marker of exposure[2]. Indeed, the author states that IgG and IgE to Stachybotrys chartarummay be the result of cross-reactivitybetween other fungi. In fact, four specimens with IgG to Stachybotrys chartarum extract were negativefor IgG to Stachyhemolysin and Stachyrase-A, and vice versa. Similar results were found for IgE. Whilethe author rightly attributes this to cross-reactivity from other fungi there is no way to determinewhether any of the reactivity he reports is Stachybotrysspecific or cross-reactivity from other fungi.We refer the author to a monograph reviewing methods for the design of epidemiologic studies with immunotoxicendpoints [4]. Third, there are several issues with the test methods and their interpretations. For example,the author states that optical density greater than 4 times the background was considered positive, whilein the Figure 2 legend he says 4 times the standard deviation is positive. Figure 2 also suggests thatboth the IgG and IgE ELISAs have the same levels of variance and mean optical density values for anti-HSAalthough the sera were diluted 1:2 for IgE and 1:100 for IgG, a finding we feel is statistically improbable.Further, in reviewing the reference cited for the isolation of Stachyrase-A [5], the authors of thatpaper state that only a short (ATQTGA) N-terminal sequence could be identified; there was difficultyin obtaining longer sequences. Did the Stachyrase-A N-terminal sequence used by Vojdani [1], containonly 6 amino acids? A simple BLAST search reveals at least 20 proteins that contain an identical sequence,none of which are Stachyrase-A. Based on the findings from the 50 subjects (patients in the Houston andLos Angeles areas) in this article and 139 blood donors (presumably from the Kansas City, MO area) [6],the authors state "It appears that about 10% of the general population and 27.4% to 42% of symptomaticmold exposed individuals have IgE antibodies to S. chartarum antigens." We don't believe this limited(both geographically and by size) meta-analysis can be representative of the general US population orwarrant such a conclusion. In closing, we feel there are significant problems with this paper which precludethe author's conclusions that antibodies to Stachyhemolysin and Stachyrase-A are new clinical biomarkersof exposure toStachybotrys chartarum. The findings and conclusions in this letter to the editor are thoseof the authors and do not necessarily represent the views of the National Institute for OccupationalSafety and Health. References: 1. Vojdani A. Antibodies against Stachybotrys chartarum extract and itsantigenic components, Stachyhemolysin and Stachyrase-A: a new clinical biomarker. Med Sci Monit, 2005;11(5): BR139-BR145 2. Trout DB, Seltzer JM, Page EH et al: Clinical use of immunoassays in assessingexposure to fungi and potential health effects related to fungal exposure. Ann All Asthma Immun, 2004;92: 483-92 3. Schmechel D, Simpson JP, Lewis DM: The production and characterization of monoclonal antibodiesto the fungus Aspergillus versicolor. Indoor Air, 2005; 15(Suppl.9): 11-19 4. Biagini RE: Epidemiologystudies in immunotoxicity evaluations. Toxicology, 1998; 129(1): 37-54 5. Kordula T, Banbula A, MacomsonJ, Travis J: Isolation and properties of stachyrase A, a chymotrypsin-like serine proteinase fromStachybotryschartarum. Infect Immun, 2002; 70(1): 419-21 6. Barnes C, Buckley S, Pacheco F, Portnoy J: IgE-reactiveproteins fromStachybotrys chartarum. Ann All Asthma Immunol, 2002; 89(1): 29-33.

Keywords: Antibodies, Fungal - blood, Antigens, Fungal, Biological Markers - blood, Hemolysin Proteins - immunology, Immunoglobulin E - blood, Immunoglobulin G - blood, Serine Endopeptidases - immunology, Stachybotrys - immunology