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Previous Article | Next Article 
Journal of Clinical Microbiology, September 2006, p. 3432-3434, Vol. 44, No. 9
0095-1137/06/$08.00+0 doi:10.1128/JCM.00856-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Dipartimento di Scienze Pediatriche e dell'Adolescenza, Università degli Studi, Turin,1 Dipartimento di Biotecnologie Cellulari ed Ematologia, Università La Sapienza, Rome, Italy2
Received 24 April 2006/ Returned for modification 22 June 2006/ Accepted 6 July 2006
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Patient 1 was a 7-year-old child with acute lymphoblastic leukemia in second relapse who underwent salvage chemotherapy in February 2004. Ten days after this treatment, the patient developed fever and periorbital edema; broad-spectrum antibiotics and liposomal amphotericin B were administered. A week later, several serum samples were positive for galactomannan antigen and caspofungin was added. A computed-tomography scan showed an abscess of the cerebral base starting from the ethmoidal plan and involving the hypophyseal region. Therefore, a surgical debridement was performed; G. capitatum was isolated from samples of pus, liquor, and bone (identification was performed with the VITEK system [bioMerieux Italia, Rome, Italy]). Galactomannan antigen was detected in the pus. Histology of a bone fragment disclosed necrotic tissue in which mycotic invasion was evident. On periodic acid-Schiff- and Grocott-stained sections, the fungi consisted of septate hyphae, slightly bent with parallel disposition and only occasional branching at a wide acute angle or budding at a right angle. A few spores and fragmentation of the mycelium in arthroconidia were also observed. Caspofungin was replaced with voriconazole. Within a few days, the patient’s clinical condition improved and galactomannan antigen, for which tests were previously constantly positive, disappeared from the blood. Two months after surgery, while under voriconazole treatment, the child underwent two consecutive aplo-identical bone marrow transplants from his father and his mother, but after a few weeks, the child died from sepsis of an unknown origin. Serum galactomannan antigen was no longer detected.
Patient 2 was a 9-year-old girl with a diagnosis of myelodysplasia secondary to acute lymphoblastic leukemia and who underwent an allogeneic bone marrow transplant, receiving bone marrow from her HLA-compatible sister. Seven months later, she developed acute myeloblastic leukemia (AML) and started intensive chemotherapy. Hematological remission was not obtained, and 3 months after AML diagnosis (April 2005), the girl underwent salvage cytotoxic treatment that resulted in a very severe and prolonged aplasia, and antifungal prophylaxis with caspofungin was started. After a week, she developed fever, a cough, and papular skin lesions. Serum galactomannan antigen was repeatedly detected at increasing concentrations, and after a week, the girl died despite broad-spectrum antibiotic and antifungal treatment with caspofungin and liposomal amphotericin B. The same day, G. capitatum was isolated from multiple blood cultures performed during the febrile period.
Patient 3 was a 49-year-old man diagnosed with AML in February 2006. After induction remission chemotherapy, he developed fever and neutropenic and antibiotic treatment with piperacillin-tazobactam was started. The fever disappeared after 48 h. Five days later, the patient was newly febrile, and a computed-tomography scan showed a pulmonary infiltrate on the left basal region associated with numerous micronodular lesions without evidence of a halo sign. Antibiotic therapy with piperacillin-tazobactam was replaced with treatment with a combination of meropenem, teicoplanin, and amikacin. Three days later, based on the persistence of fever and the appearance of multiple little papular skin lesions highly suggestive of candida infection, treatment with liposomal amphotericin B (5 mg/kg/day) was started. A serum sample was positive for galactomannan antigen, and cultures from blood and from a skin lesion needle aspirate yielded a yeast morphologically compatible with G. capitatum. Based on the previous experience with the two above-described cases of G. capitatum infection, liposomal amphotericin B was replaced with voriconazole. Two days later, identification of the G. capitatum yeast was confirmed with the VITEK system and a second serum sample was positive for galactomannan antigen. After a week of voriconazole therapy, the patient developed a respiratory failure and died despite broad-spectrum antibiotic and antifungal treatment. A bronchoalveolar lavage sample was negative for fungi and mycobacteria but yielded Stenotrophomonas maltophilia. Two further serum samples were negative for galactomannan antigen.
Despite the positive Platelia Aspergillus assay results for multiple samples from the three patients, the histopathological, microbiological, and clinical findings suggested that coinfection by G. capitatum and Aspergillus spp. was unlikely. However, the possibility of an undetected Aspergillus infection cannot be formally excluded because the patients did not undergo autoptic examination and Aspergillus PCR was not performed on the available specimens. At the time of serum sample collection, the patients did not receive antibiotics, such as piperacillin-tazobactam or amoxicillin-clavulanate, known to cause false positivity in the Platelia Aspergillus assay (11). Therefore, we hypothesized that Platelia Aspergillus positivity may have been due to a cross-reaction with antigens released by G. capitatum. Consequently, we tested 17 clinical strains of G. capitatum and other control fungi for their reactivity in the ELISA assay, according to a previously described procedure (4) (Table 1). Cultures were plated on Sabouraud dextrose agar and incubated at 30°C for 5 days. For each strain, 5 to 10 colonies were suspended in 1 ml distilled water. After vigorous agitation, the suspensions were centrifuged for 5 min at 10,000 x g, and the supernatants were heated and tested with Platelia Aspergillus. Each experiment included several controls, i.e., distilled water and negative, positive, and standard (1-ng/ml) serum samples provided by the manufacturer. The ratio between the optical density of the serum sample and that of the control standard serum was calculated for each sample. According to the recently suggested 0.5 cutoff value for the test, values of 
0.5 were considered relevant (10). The 17 G. capitatum strains, including the isolate from patient 3 (isolates from patients 1 and 2 were no longer available), showed various positive indexes of reactivity (range, 1.2 to >5). The three Aspergillus strains and the two strains of Cryptococcus neoformans showed high and intermediate indexes of reactivity, respectively, in the Platelia Aspergillus assay. The two reference Candida strains did not show any reactivity.
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Our three cases and in vitro studies show that G. capitatum exoantigens may be cross-reactive in the Aspergillus galactomannan assay. Although almost all of the yeast mannans do not contain other carbohydrate components except a trace of N-acetylglucosamine and glucose, some yeasts, including Geotrichum spp., are known to have galactomannan as a cell wall component (1), but until now, no data on detection of circulating galactomannan in patients with invasive infection due to this genus of yeasts have been published.
G. capitatum, formerly known as Trichosporon capitatum or Blastoschizomyces capitatus, is an uncommon, but frequently fatal, cause of invasive infections in immunocompromised patients, particularly those with hematological malignancies (6). In a recent retrospective multicenter study from Italy, the incidence of G. capitatum infection among patients with acute leukemia was 0.5%, with a 55.7% crude mortality rate (6). Early diagnosis is difficult to establish because clinical symptoms are not specific and frequently resemble those of invasive candidosis. Blood cultures, which are the "gold standard" for diagnosis of invasive G. capitatum infections, are negative in about 30% of cases and usually take several days to show detectable growth of yeast cells. Therefore, noncultural methods could be useful for the improvement of laboratory diagnosis, but experiments on G. capitatum antigens or DNA detection are not available to date. This false-positive reactivity in the Platelia Aspergillus assay does not seem to constitute an important problem for specificity in the diagnosis of invasive aspergillosis, as the incidence of G. capitatum infections is very low even in leukemic patients (6). On the contrary, this unexpected reactivity in the test could be considered a useful diagnostic and prognostic tool in the management of G. capitatum infections. A positive galactomannan assay result for a patient with an infective clinical picture suggestive of Candida infection could indicate the possibility of a G. capitatum infection, considering also that galactomannan serum detection may precede by several days the microbiological documentation of the infection (for patients 1, 2, and 3, serum galactomannan was detected 7, 9, and 2 days before identification of the pathogen from cultures, respectively). This is a crucial point not only for specific diagnosis but also for the choice of antifungal therapy. In fact, G. capitatum is susceptible to amphotericin B and azoles, in particular voriconazole, but is intrinsically resistant to echinocandins, which are drugs of choice for the treatment of invasive Candida and Aspergillus infections (6). Interestingly, for patient 1, infection improved when caspofungin was replaced by voriconazole; patient 2 developed a breakthrough G. capitatum infection while under antifungal prophylaxis with caspofungin; and for patient 3, the choice of voriconazole therapy was based on the observation of multiple maculopapular skin lesions suggestive of disseminated Candida infection, the positivity of the Platelia Aspergillus assay, and the experience with the previous two cases of G. capitatum infection.
In conclusion, the cases we have presented and in vitro tests showed a previously unknown cross-reactivity of G. capitatum with Aspergillus galactomannan. Although this cross-reactivity may constitute a drawback for the specificity of the test, the knowledge of this phenomenon may be useful in the diagnosis and management of invasive G. capitatum infections, which are rare but cause severe complications in patients with hematological malignancies.
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