Assessment of Aspergillus-Specific T Cells for Diagnosis of Invasive Aspergillosis in a Leukemic Child with Liver Lesions Mimicking Hepatosplenic Candidiasis

ABSTRACT A child with acute myeloid leukemia presented with multiple liver lesions mimicking hepatosplenic candidiasis during the neutropenic phase following the induction chemotherapy. All the available diagnostic tools showed repeatedly negative results, including galactomannan. An enzyme-linked immunospot (ELISPOT) assay showed a high number of Aspergillus-specific T cells producing interleukin-10 [TH2(IL-10)] and a low number of Aspergillus-specific T cells producing gamma interferon [TH1(IFN-γ)], revealing invasive aspergillosis (IA) before the confirmatory biopsy. A progressive skewing from the predominance of TH2(IL-10) to a predominance of TH1(IFN-γ) was observed close to the complete resolution of the infection and foreshadowed the outcome. The ELISPOT assay holds promise for diagnosing pediatric IA.

On day 9 of induction chemotherapy (10 mg/m 2 idarubicin, days 1 to 3; 200 mg/m 2 cytarabine, days 1 to 7; 100 mg/m 2 etoposide, days 1 to 5), an 11-year-old child with acute myeloid leukemia (AML) presented with febrile neutropenia. A complete cultural and molecular examination (CCME) of blood, urine, and stool was repeatedly unrevealing. The galactomannan antigenemia (GM) test, performed twice a week, and the complete pulmonary radiological study (PRxS) results were negative. Empirical antibiotic therapy was ineffective, and empirical intravenous fluconazole (12 mg/kg of body weight/day) was undertaken. On day 28, the febrile patient presented with pain in the right flank and abdominal tenderness. Laboratory tests showed neutrophil recovery, a slight increase in levels of transaminases, and a marked increase of alkaline phosphatase. On day 33, an abdominal ultrasound (AUS) disclosed multiple hypoechoic liver lesions, with sparse hyperechoic features (Fig.  1A). PRxS results remained negative. Fluconazole was replaced by liposomal amphotericin B (L-AMB) (1.5 mg/kg endovenously), with improvement of the symptoms. On day 42, the patient, showing complete remission of AML, underwent a second chemotherapeutic course with the same schedule. Con-comitantly, the lesions persisted without mutations as determined by AUS. On day 45, the fever again rose above 38°C. CCME and GM results remained negative. Empirical antibiotic therapy was reintroduced, and L-AMB was increased to 3 mg/kg of body weight/day. On day 51, AUS revealed a slight reduction in the sizes of the liver lesions. Nonetheless, fever persisted until bone marrow recovery. On day 62, a third course was undertaken (cytarabine, 3 g/m 2 twice a day, days 1 to 3; etoposide, 125 mg/m 2 , days 2 to 5). On day 69, after a further febrile episode, AUS displayed an enlargement of the liver lesions (Fig. 1B). CCME and GM results continued to be negative. We judged it appropriate to explore the patient's Aspergillus-specific T-cell responses by a recently described application of an enzyme-linked immunospot (ELISPOT) assay to definitely rule out invasive aspergillosis (IA) (9). On day 75, the peripheral blood sample collected for the ELISPOT assay was positive for a high number of Aspergillus-specific T cells producing interleukin-10 [TH2(IL-10)] and for a low number of Aspergillus-specific T cells producing gamma interferon [TH1(IFN-␥)] (Fig. 1G). On day 80, a liver biopsy disclosed hepatic IA by Aspergillus fumigatus upon either a histologic ( Fig. 2A and B) or a cultural (not shown) examination. Caspofungin (50 mg/day) was associated with L-AMB. On day 84, an ELISPOT assay confirmed a very high number of TH2(IL-10) and a low number of TH1(IFN-␥) (Fig. 1G). On day 95, the fever resolved. On day 102, an AUS showed a slight decrease in the sizes of the liver lesions (Fig. 1C). Eight days later, an ELISPOT assay demonstrated a sustained number of TH2(IL-10) and a stable number of TH1(IFN-␥)  ϫ200) showed an extensive granulomatous inflammation of the liver with dispersed birefringence filamentous structures (inset magnification, ϫ400). (B) Silver staining (magnification, ϫ200) showed septate, acutely branching, hyphae in a pattern typical of aspergillosis (inset magnification, ϫ400).

VOL. 15, 2008
CASE REPORTS 1627 (Fig. 1G). A 3-g/m 2 sample of cytarabine twice a day (days 1 to 3) and 10 mg/m 2 mitoxantrone (days 3 and 4) were administered without additional complications. On day 132, because of a smaller reduction in the sizes of the liver lesions as determined by AUS (Fig. 1D), L-AMB was withdrawn. On day 166, an ELISPOT assay continued to be positive, with a decreased number of TH2(IL-10) and a steady number of TH1(IFN-␥) (Fig. 1G). Four days later, an AUS revealed a significant reduction in the sizes and number of the liver lesions (Fig. 1E). On day 190, 4 g/m 2 cyclophosphamide and 200 mg/m 2 etoposide (days 2 to 4) were given. The neutropenia elapsed asymptomatically. On day 204, the ELISPOT result was positive only for TH1(IFN-␥) (Fig. 1G). On day 219, the AUS revealed a homogenous liver without focal lesions, and caspofungin was finally discontinued (Fig. 1F). The patient is well and in complete remission.
IA and invasive candidiasis represent the most common causes of infection-related mortality in pediatric patients with AML (11). Data from the Centers for Disease Control and Prevention show that the mortality associated with IA has increased 357% since 1980, due to the limitation of diagnostic tools in establishing a prompt and undoubted diagnosis (7). Also, testing for GM, the most widely used noncultural method, is hampered by having a variable diagnostic accuracy, especially for children (2,8).
Clinically, pulmonary infiltrates are the most common presentation of IA, with liver involvement occurring in less than 15% of patients and only in the late phase of the infection (3). In contrast, visceral presentation is common with invasive candidiasis, especially in the chronic form, which is also termed hepatosplenic candidiasis (HSC). HSC occurs in 3% to 7% of neutropenic patients and persists for months, despite granulocyte recovery and antifungal treatment. Fever, upper gastrointestinal symptoms, and increased serum alkaline phosphatase characterizes HSC, and it has four dominant ultrasonographic patterns: "wheel within a wheel," "bull's eye," hypoechoic lesion, and echogenic foci (4).
Only four cases of primary liver IA have so far been reported; they have occurred in three adult patients and one pediatric patient, who presented with either a single lesion or multiple lesions. In all four patients, the diagnosis was proven by biopsy and preceded by a single positive GM test in only one case (5,6,12,13). This is a further case of liver IA mimicking HSC on clinical and radiological grounds. Of note, while all the other diagnostic tools, including GM testing, showed repeatedly negative results, a positive ELISPOT result provided the sole hint of IA and shortly preceded the confirmatory biopsy. We recently reported the potential of the ELISPOT assay to diagnose IA in adult patients with pulmonary infiltrates (9). Also, in this case of extrapulmonary IA, the ELISPOT assay provided the kinetics of the Aspergillus-specific T-cell responses during the course of IA by showing (i) a permissive response, polarized in TH2(IL-10), at the onset; (ii) a stable TH1(IFN-␥) response with a progressive decrease of the TH2(IL-10) response, which in turn was sufficient to counterbalance the TH1(IFN-␥) and avoid an excessive inflammatory response during the regression phase; and (iii) a protective response, polarized in TH1(IFN-␥), at the complete resolution (Fig. 2). This is reminiscent of the kinetics of the Aspergillus-specific T-cell responses in the mouse model of IA and consistent with the notion that the presence of Aspergillus-specific TH1(IFN-␥) responses provides a better chance for stem cell transplant patients to survive IA (1,10).
In conclusion, liver IA should be considered in the differential diagnosis of liver lesions in neutropenic patients, as should HSC, tuberculosis, and staphylococcal infections. The ELISPOT assay may have a role in the diagnosis of IA in pediatric patients as well. The diagnostic accuracy of the ELISPOT assay needs larger confirmatory studies.