Galactomannan enzymatic immunoassay cross-reactivity caused by. Prototheca species

Transcription

1 JCM Accepts, published online ahead of print on 25 July 2012 J. Clin. Microbiol. doi: /jcm Copyright 2012, American Society for Microbiology. All Rights Reserved Galactomannan enzymatic immunoassay cross-reactivity caused by Prototheca species Van den Bossche D. 1, De Bel A. 1*, Hendrickx M. 2, De Becker A. 3, Jacobs R. 4, Naessens A. 1, Piérard D. 1 1 Department of Microbiology and Infection Control, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, 1090 Brussels, Belgium. 2 BCCM/IHEM collection, Mycology and Aerobiology Section, Scientific Institute of Public Health, Juliette Wytsmanstraat 14, 1050 Brussels, Belgium. 3 Department of Haematology, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, 1090 Brussels, Belgium. 4 Department of Intensive Care, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, 1090 Brussels, Belgium. *Corresponding author. Mailing address: Department of Microbiology and Infection Control, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, 1090 Brussels, Belgium. Phone: Fax: Annelies.DeBel@uzbrussel.be Running title: Galactomannan cross-reactivity by Prototheca species 21 1

2 Abstract We report a reactive Aspergillus galactomannan enzymatic immunoassay against the serum of a patient with invasive Prototheca zopfii infection. Analysis of the supernatants of suspensions of P. zopfii and other Prototheca isolates revealed positive results as well. These data suggest cross-reactivity with the serum Aspergillus galactomannan assay in invasive protothecosis. Main text Prototheca species are classified as achlorophyllic algae and are opportunistic pathogens (7). Prototheca infections are generally divided into three groups: olecranon bursitis, localized cutaneous infections and disseminated disease. The latter is more commonly reported in severely immunocompromised individuals, whereas localized infections can present in patients with normal host immunity (9). Therapeutic recommendations for protothecal infections are limited because susceptibility profiles are variable and do not correlate with successful treatment (8). Generally, treatment consists of medical and/or surgical therapy. Based on data from several case reports and in vitro studies, amphotericin B is presumably the most effective therapy (8-9). Correct identification of Prototheca species is of therapeutic and prognostic value but may be difficult due to their yeast-like colony appearance on routine media (14). Here, we report on another possible misleading aspect of invasive protothecal infections. We describe the case of a 63-year-old man diagnosed with follicular B-cell non- Hodgkin s lymphoma, stage IV in Following two relapses (2007 and 2010), autologous (2008) and allogeneic stem cell (2010) transplantation were performed. 2

3 Subsequently, he developed chronic graft versus host disease (GVHD) in the summer of 2011, which was treated with cyclosporin and methylprednisone. Because the patient was unable to ingest sufficient food and did not tolerate posaconazole antifungal prophylaxis, as recommended by the European Conference on Infections in Leukemia (ECIL) guidelines (13), fluconazole was administered. Three months later, during a routine follow-up consultation, serum galactomannan (Platelia, Bio-Rad Inc., Marnes-la- Coquette, France) revealed a positive index of 0.58 (normal value: index < 0.50). Fluconazole was replaced by posaconazole, as the patient s food intake was improved. Five days later, the patient was admitted to the intensive care unit with acute coronary syndrome. Chest X-ray and high resolution computed tomography (CT) showed a bilateral interstitial infiltrate and pulmonary congestion. A follow-up sample, taken six days after the first positive sample, confirmed galactomannan positivity with an index of 2.31, and treatment was switched to lipid complex amphotericin B. At hospital admittance, two out of three aerobic blood cultures were positive, and growth on blood and Sabouraud agar (BD Difco Laboratories, Le Pont De Claix, France) revealed yeast-like colonies. Pink colonies were observed on Candida CHROMagar (CHROMagar Company, Paris, France). Both unstained and calcofluor stained wet mount of the colonies showed characteristic asymmetrical morula-like structures. Protothecal infection was suspected, but the API 20C AUX (biomérieux, Marcy l Etoile, France) assimilation profile did not allow identification. Furthermore, identification could not be obtained with matrix-assisted laser-desorption/ionization timeof-flight mass spectrometry (Microflex, Bruker Daltonik GmbH, Bremen, Germany, database version ). Finally, identification of Prototheca zopfii by 18S rrna 3

4 sequencing was established by the Belgian Coordinated Collections of Micro-Organisms/ Institute of Hygiene and Epidemiology, Mycology (now Scientific Institute of Public Health) (BCCM/IHEM). Two follow-up aerobic blood cultures taken two days after admittance were still positive. Treatment with amphotericin B was continued with an initial good clinical response, and additional blood cultures were negative. Nevertheless, two weeks later, the patient s condition deteriorated, and he died. No autopsy was performed. During these two weeks, serum galactomannan antigen was repeatedly positive (Figure 1), and bronchoalveolar lavage (BAL) showed a galactomannan index of The normal value for BAL samples is currently debated (4), but this result should be considered positive. Despite the repeatedly positive galactomannan results, conventional diagnostic methods for Aspergillus infection gave negative results: no Aspergillus species could be cultured, and no radiographic patterns compatible with the European Organization for Research and Treatment of Cancer/ Mycosis Study Group (EORTC/MSG) definitions (3) were seen. However, in GVHD patients, elevated galactomannan levels due to translocation of dietary galactomannan through the intestinal mucosa into the blood are described (1,6,11). Other possible known causes of false-positive galactomannan results were excluded (12,16,19). Because co-infection with P. zopfii and Aspergillus spp. was rather unlikely, cross-reactivity in the Platelia Aspergillus assay caused by Prototheca was suspected. To investigate the occurrence of reactivity in the serum galactomannan assay caused by Prototheca species, we retrospectively analyzed a serum sample of a patient recently 4

5 diagnosed in our laboratory with P. wickerhamii infrapatellar bursitis (21). The serum sample from this patient did not show reactivity in the galactomannan assay (index 0.11). The infection, however, was limited to the knee and not disseminated. Subsequently, we analyzed supernatants from these two strains (P. zopfii and P. wickerhamii) and three additional strains, which were selected from the BCCM/IHEM collection (P. zopfii IHEM 20268, P. stagnora IHEM 4201 and P. cutis IHEM 23764). Distilled water, a Candida albicans clinical isolate (negative control) and an Aspergillus fumigatus clinical isolate (positive control) were included as controls. Using the conditions described by Dalle et al. (2), suspensions of three isolates - P. zopfii clinical isolate, P. wickerhamii clinical isolate and P. stagnora IHEM resulted in high galactomannan assay index values of 3.29, 1.92 and 1.77, respectively. For the two other strains - P. cutis IHEM and P. zopfii IHEM only weak reactivity (indexes between 0.53 and 1.06) was found when heavy suspensions were used. These results show that Prototheca spp. can cause reactivity in the galactomannan assay and suggest that the positive serum and BAL galactomannan tests in the patient with invasive infection are caused by Prototheca, although a co-infection with Aspergillus could not be excluded. The serum galactomannan enzymatic immunoassay is widely used for the diagnosis of aspergillosis in immunocompromised hosts. Since its introduction, some false positive results caused by cross-reactivity with other fungi and yeasts, such as Cryptococcus neoformans, Geotrichum capitatum, Penicillium spp., Alternaria spp., Histoplasma spp. and Paecilomyces spp., have been demonstrated (2,5,18,20,22). To the best of our 5

6 knowledge, no data are available on the detection of galactomannan antigen in serum from patients with invasive protothecosis. Galactomannan is not reported to be present in the cell wall of Prototheca species. P. zopfii β-glucan consists of a 1,4-linked (90%) and 1,3-linked (10%) glucose backbone (17), whereas Aspergillus galactomannan has a 1,4- linked mannose basic structure with galactose branching in position 6. However, comparison of the monosaccharide cell wall composition of Aspergillus niger and P. zopfii illustrated the presence of glucose and mannose in both species but the absence of galactose in P. zopfii (10). It is unclear how cross-reactivity originated. The molecule responsible for cross-reactivity might be a structural component of the cell wall or a secreted glycoprotein. The impact of this newly detected cross-reactivity is expected to be limited, as invasive protothecosis has a low prevalence (9). However, differentiation between Aspergillus and Prototheca is important for the choice of antifungal therapy: echinocandins can be used to treat invasive aspergillosis, whereas no data are available for their activity against Prototheca spp. (15). This report confirms once more that positive galactomannan levels should be interpreted with caution. Both clinical Prototheca strains were deposited in the BCCM/IHEM collection (P. wickerhamii: IHEM and P. zopfii: IHEM 25445). The 18S rrna sequence for P. zopfii was deposited in Genbank (JQ679396)

7 Acknowledgments Possible conflicts of interest are that we receive an annual funding from Pfizer for the performance of the galactomannan assay

8 References 1. Ansorg, R., R. van den Boom, P.M. Rath Detection of Aspergillus galactomannan antigen in foods and antibiotics. Mycoses. 40: Dalle, F., P.E. Charles, K. Blanc, D. Caillot, P. Chavanet, F. Dromer, and A. Bonnin Cryptococcus neoformans galactoxylomannan contains an epitope's that is cross-reactive with Aspergillus galactomannan. J. Clin. Microbiol. 43: De Pauw, B., T.J. Walsh, P. Donnelly, D.A. Stevens, J.E. Edwards, T. Calandra, P.G. Pappas; J. Maertens, O. Lortholary, C.A. Kauffman, D.W. Denning, T.F. Patterson, G. Maschmeyer, J. Bille, W.E. Dismukes, R. Herbrecht, W.W. Hope, C.C. Kibbler, B.J. Kullberg, K.A. Marr,P. Muñoz, F.C. Odds, J.R. Perfect, A. Restrepo, M. Ruhnke, B.H. Segal, J.D. Sobel, T.C. Sorrell, C. Viscoli, J.R. Wingard, T. Zaoutis, and J.E. Bennett Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/ Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin. Infect. Dis. 46: D Haese J., K. Theunissen, E. Vermeulen, H. Schoemans, G. De Vlieger, L. Lammertijn, P. Meersseman, W. Meersseman, K. Lagrou, and J. Maertens. 1 February Galactomannan detection in bronchoalveolar lavage fluid of patients at risk of invasive pulmonary aspergillosis: analytical and clinical validity. J. Clin. Microbiol. doi: /jcm

9 Giacchino, M., N. Chiapello, S. Bezzio, F. Fagioli; P. Saracoo, A. Alfarano, V. Martini, G. Cimino, P. Martino, and C. Girmenia Aspergillus galactomannan enzyme-linked immunosorbent assay cross-reactivity caused by invasive Geotrichum capitatum. J. Clin. Microbiol. 44: Hamaki, T., M. Kami, Y. Kanda, S. Miyakoshi, J. Ueyama, S. Morinaga, and Y. Mutou False-positive results of Aspergillus enzyme-linked immunosorbent assay in a patient with chronic graft-versus-host disease after allogeneic bone marrow transplantation. Bone Marrow Transplant. 28: Huerre, M., P. Ravisse, H. Solomon, P. Ave, N. Briquelet, S. Maurin, and N. Wuscher Human protothecosis and environment. Bull. Soc. Pathol. Exot. 86: Iacoviello, V.R., P.C. DeGirolami, J. Lucarini, K. Sutker, M.E. Williams, and C.A. Wanke Protothecosis complicating prolonged endotracheal intubation: case report and literature review. Clin. Infect. Dis. 15: Lass-Flörl, C., and A. Mayr Human protothecosis. Clin. Microbiol. Rev. 20: Lloyd, D., and G. Turner The cell wall of Prototheca zopfii. J. Gen. Microbiol. 50: Maertens, J., J. Verhaegen, H. Demuynck, P. Brock, G. Verhoef, P. Vandenberghe, J. Van Eldere, L. Verbist and M. Boogaerts Autopsycontrolled prospective evaluation of serial screening for circulating galactomannan by a sandwich enzyme-linked immunosorbent assay for 9

10 haematological patients at risk for invasive aspergillosis. J. Clin. Microbiol. 37: Maertens, J., K. Theunissen, G. Verhoef and J. Van Eldere Falsepositive Aspergillus galactomannan antigen test results. Clin. Infect. Dis. 39: Maertens, J., O. Marchetti, R. Herbrecht, O.A. Cornely, U. Flückiger, P. Frêre, B. Gachot, W.J. Heinz, C. Lass-Flörl, P. Ribaud, A. Thiebaut, and C. Cordonnier European guidelines for antifungal management in leukemia and hematopoietic stem cell transplant recipients: summary of the ECIL Update. Bone Marrow Transplant. 46: McMullan, B Prototheca wickerhamii mimicking yeast: a cautionary tale. J. Clin. Microbiol. 49: Narita, M., R.R. Muder, T.V. Cacciarelli, and N. Singh Protothecosis after liver transplantation. Liver Transpl. 14: Racil, Z., I. Kocmanova, M. Lengerova, J. Winterova, and J. Mayer Intravenous Plasma-Lyte as a major cause of false-positive results of Platelia Aspergillus test for galactomannan detection of serum. 45: Rivas, L.A., and R. Pont Lezica Synthesis of β-glucans in Prototheca zopfii. Isolation and characterization of the glycoprotein primer. Eur J. Biochem. 163: Stynen, D., J. Sarfati, A. Goris, M.C. Prevost, M. Lesourd, H. Kamphuis, V. Darras, and J. P. Latge Rat monoclonal antibodies against Aspergillus galactomannan. Infect. Immun. 60:

11 Sulahian, A., S. Touratier, and P. Ribaud False positive test for Aspergillus antigenemia related to concomitant administration of piperacillin and tazobactam. N. Eng. J. Med. 24: Swanink, C.M.A., J.F.G.M. Meis, A.J.M.M. Rijs, J.P. Donnelly, and P.E. Vreweij Specificity of a sandwich enzyme-linked immunosorbent assay for detecting Aspergillus galactomannan. J. Clin. Microbiol. 35: Van den Bossche D., R. de Haan, J. Van der Werff ten Bosch, W. Van Hecke, F. Symoens, I. Van den Borre, S. Allard, A. De Bel. Case Report: Infrapatellar bursitis caused by Prototheca wickerhamii. Medical Mycology Case Reports, in press. 22. Wheat, L.J., E. Hackett, M. Durkin, P. Conolly, R. Petraitiene, T.J. Walsh, K. Knox, C. Hage Histoplasmosis-associated cross-reactivity in the Bio- Rad PlateliaTM Aspergillus enzyme immunoassay. Clin. Vaccine Immunol. 14:

12 FIG. 1. Evolution of serum galactomannan index over time for the patient with invasive protothecosis. Threshold for positivity (0.50) is indicated by the dashed line. Day 0: last negative galactomannan index two weeks prior to hospital admission. Dashed arrow: initiation of posaconazole. Bold arrow: hospital admission with change to amphotericin B treatment. 12

13