The Platelia Aspergillus antigen enzyme-linked immunosorbent assay (ELISA) (Bio-Rad, Marnes-la-Coquette, France) has become an important tool for the early detection of invasive aspergillosis in immunocompromised patients (4). This ELISA uses a rat monoclonal antibody (EB-A2) which reacts with β-(1,5)-galactofuranose, a side chain of galactomannan (GM). Cross-reactivity of the Platelia ELISA with fungi other than Aspergillus was reported previously (2). Over 10 years ago, we discovered cross-reactivity of GM in antibiotics of fungal origin, such as ampicillin-sulbactam and piperacillin, by the Pastorex Aspergillus latex agglutination test, which used the same antibody (1). Industrially produced β-lactam antibiotics derived from Penicillium species also showed positive GM values in vivo (5).
The echinocandins inhibit the synthesis of glucan in the cell walls of several molds and are effective for treatment of candidemia. In addition, caspofungin is recommended for salvage therapy in invasive aspergillosis.
Echinocandins are sterile lyophilized agents for intravenous infusion that contain semisynthetic lipopeptide compounds synthesized from fermentation products of different fungi. Pneumocandin B, the semisynthetic derivative of caspofungin, is manufactured from the fungus Zalerion arboricola, anidulafungin from Aspergillus nidulans, and micafungin from Coleophoma empetri. These three filamentous fungi belong to the phylum Ascomycota. Aspergillus and Penicillium, which contain galactomannoproteins in the cell wall, also belong to this phylum.
The purpose of this study was to detect the presence of GM in preparations of industrially produced echinocandins by the Aspergillus antigen ELISA. In addition, the GM values of the supernatants of the ingredient fungi were tested by culturing them on Sabouraud agar at 37°C for 2 days. Broth cultures were obtained by inoculating 1 cm3 mature mycelia into 20 ml of the GM-negative isotonic solution (sterofundin). The isolates were incubated at room temperature for 5 days, and the fungal supernatant was decanted; 300 μl of the supernatant was used in the Aspergillus antigen assay.
All batches of Cancidas (caspofungin) infusion solutions tested were GM positive (cutoff index in serum is currently 0.5), whereas GM detection in Ecalta/Eraxis (anidulafungin) was variable in different batches and Mycamine (micafungin) gave negative test results (Table 1). The median GM indices (n = 3) were 4.4, 0.8, 0.65, <0.5, and <0.5 for 70 mg and 50 mg caspofungin, 100 mg anidulafungin, and 50 mg and 100 mg micafungin, respectively. In the supernatants of the fungi used for manufacturing the echinocandins, GM indices were also detected.
TABLE 1.
Galactomannan (GM) values of antifungal infusion solutions by blind testinga
| Drug name | Drug concn (mg) | Qualitative resultb | GM index of antifungal infusion solution |
GM index of fungal supernatant solutionc | |
|---|---|---|---|---|---|
| Median | Range | ||||
| Caspofungin (Cancidas) | 70 | Positive | 4.4 | 3.1-5.2 | 4.0 (Zalerion arboricola) |
| 50 | Positive | 0.8 | 0.5-1.0 | ||
| Anidulafungin (Ecalata) | 100 | Positive | 0.65 | 0.1-1.8 | 6.1 (Aspergillus nidulans) |
| Micafungin (Mycamine) | 50 | Negative | <0.5 | <0.5 | 3.7 (Coleophoma empetri) |
| 100 | Negative | <0.5 | <0.5 | ||
Three batches were tested for each drug and each concentration.
The GM index was estimated by the ratio of the absorbance value to the cutoff value and considered positive when the value was ≥0.5 on repeat testing.
GM index of the supernatant solution from the ingredient fungus. The fungal species is shown in parentheses.
Due to the GM content of caspofungin, especially in the 70-mg dose which is used as the loading dose, the detection of circulating GM may be influenced during caspofungin administration. It is important to point out that the concentrations of GM in caspofungin infusion solutions do not reflect the concentrations of GM circulating in the bloodstream. Nevertheless, increases of GM levels have been reported in vivo and in vitro following echinocandin exposure (3). To our knowledge, this is the first report of GM detection in echinocandin preparations. These findings can have meaningful impact for interpretation of GM antigenemia in patients receiving echinocandins, especially caspofungin. Clinical studies are necessary to verify the relevance of positive serum GM levels and concomitant caspofungin administration.
Acknowledgments
We thank J. Dedy from the Pharmacy Department of the University Hospital Essen for providing different batches of echinocandins.
Footnotes
Published ahead of print on 31 March 2010.
REFERENCES
- 1.Ansorg, R., R. van den Boom, and P. M. Rath. 1997. Detection of Aspergillus galactomannan antigen in foods and antibiotics. Mycoses 40:353-357. [DOI] [PubMed] [Google Scholar]
- 2.Cummings, J. R., G. R. Jamison, J. W. Boudreaux, M. J. Howles, T. J. Walsh, and R. T. Hayden. 2007. Cross-reactivity of non-Aspergillus fungal species in the Aspergillus galactomannan enzyme immunoassay. Diagn. Microbiol. Infect. Dis. 59:113-115. [DOI] [PubMed] [Google Scholar]
- 3.Klont, R. R., M. A. Mennink-Kersten, D. Ruegebrink, A. J. Rijs, N. M. Blijlevens, J. P. Donnelly, and P. E. Verweij. 2006. Paradoxical increase in circulating Aspergillus antigen during treatment with caspofungin in a patient with pulmonary aspergillosis. Clin. Infect. Dis. 43:e23-e25. [DOI] [PubMed] [Google Scholar]
- 4.Maertens, J., J. Verhaegen, K. Lagrou, J. Van Eldere, and M. Boogaerts. 2001. Screening for circulating galactomannan as a noninvasive diagnostic tool for invasive aspergillosis in prolonged neutropenic patients and stem cell transplantation recipients: a prospective validation. Blood 97:1604-1610. [DOI] [PubMed] [Google Scholar]
- 5.Sulahian, A., S. Touratier, and P. Ribaud. 2003. False positive test for Aspergillus antigenemia related to concomitant administration of piperacillin and tazobactam. N. Engl. J. Med. 349:2366-2367. [DOI] [PubMed] [Google Scholar]