INTRODUCTION

TOP ABSTRACT INTRODUCTION DISCUSSION REFERENCES

Patients with chronic granulomatous disease (CGD) are at risk for fungal infections such as invasive aspergillosis. Although the course of infection is usually more protracted than in patients with neutropenia, treatment is often extremely difficult because the underlying disease cannot be resolved. Here we describe a case of invasive Aspergillus nidulans infection with chest-wall invasion in a patient with CGD in whom treatment with amphotericin B failed , but who showed a favorable response to treatment with a new antifungal triazole, voriconazole.

	CASE REPORT

A male child was born in 1991 to nonconsanguinous parents after an uncomplicated pregnancy. CGD was diagnosed at 22 mo of age. Because DNA analysis excluded X-linked CGD phox⁴⁷ and phox⁶⁵, the patient presumably had CGD phox²². At nearly 5 yr of age (weight 15 kg) he started coughing and became tachypneic, with signs of left-sided pneumonia. A chest X-ray confirmed the presence of a left-sided pulmonary consolidation, and the patient was treated orally with amoxicillin/clavulanic acid followed by cefuroxim-axetil without clinical improvement. Because chest X-rays showed progression of the pulmonary infiltrates, bronchoalveolar lavage (BAL) was performed. Direct microscopy revealed septate hyphae with dichotomous branching at acute angles that is suggestive of Aspergillus species, but cultures remained sterile. Itraconazole capsules (6 mg/kg/d; Janssen-Cilag B.V., Tilburg, The Netherlands) were administered under the suspicion of invasive pulmonary aspergillosis. Two days later the patient was readmitted with a cutaneous abscess on his back, which was punctured. Culture of this abscess grew only Aspergillus nidulans. Intravenous amphotericin B-desoxycholate (Bristol-Myers Squibb B.V., Woerden, The Netherlands), at a dose of 1 mg/kg/d, was added to the regimen. A computed tomogram (CT) of the thorax and abdomen showed left-sided basal pneumonic infiltrates, a left-sided subdiaphragmal fluid collection, splenomegaly, and hepatomegaly with a perihepatic fluid collection without radiologic signs of liver abscesses. There was no evidence of pericardial effusion. One week after admission of the patient, enteral feeding was no longer tolerated, and itraconazole had to be stopped (cumulative dose: 42 mg/kg). Interferon- (IFN-) (Boehringer Ingelheim B.V., Alkmaar, The Netherlands), in a dose of 35 µg subcutaneously three times weekly (1), and filgrastim (Roche Nederland B.V., Mijdrecht, The Netherlands) in a dose of 100 µg subcutaneously once daily (2), were added to the patient's regimen, but had to be discontinued because of side effects. In addition, five granulocyte transfusions from human leukocyte antigen (HLA)-related donors were given (3), but these had to be stopped because of anaphylactic shock. Donor neutrophils showed excellent in vitro oxidative activity against Staphylococcus aureus and against an A. nidulans isolate cultured from the patient's cutaneous abscess, but the neutrophil oxidative activity decreased rapidly, and only 0.01% of the baseline oxidative activity could be detected in the patients' blood 48 h after the granulocyte transfusion. After three of five granulocyte transfusions the patient's clinical condition deteriorated further, and progressive respiratory insufficiency and fluid retention were observed. Echocardiography revealed pericardial effusion with signs of incipient heart tamponade, and the patient was transferred to the pediatric intensive care unit (ICU). Intubation was performed and mechanical ventilation was started, and immediate surgical drainage of the pericardial effusion was done. Culture and histologic examination of pericardial fluid and a pericardial biopsy specimen were unrevealing. Surgical exploration of the ventral cutaneous abscess showed it to be an intrathoracic abscess without subphrenic connection. Cultures still grew A. nidulans. Therapy was continued with local instillation of healthy allogenic granulocytes twice daily (4). Despite 6 wk of treatment with amphotericin B (cumulative dose: 60 mg/kg) combined with immunomodulative therapy, the patient showed an increased need for ventilatory and inotropic support, and a repeat CT of the thorax and abdomen showed progressive bilateral pulmonary infiltrates. Since the patient's condition had not improved with the existing treatment regimen, the amphotericin B was discontinued and intravenous therapy was begun with voriconazole (Pfizer Central Research, Sandwich, UK) after informed consent was obtained from the patient's parents. The dose and blood levels of voriconazole are shown in Figure 1. After 4 wk of intravenous treatment with voriconazole, the patient's clinical condition improved, confirmed by follow-up CT, which showed regression of pulmonary infiltrates. Extubation was achieved 43 d after admission to the ICU. Intravenous voriconazole was continued for a total of 10 wk and then changed to oral therapy (7 mg/kg, twice daily). The drug was tolerated well, and no adverse events were observed during treatment. The patient was discharged to go home while receiving this medication, and there was a steady clinical improvement in his condition over the following months. Voriconazole was stopped after 8¹/₂ mo of therapy, and antifungal prophylaxis was continued with itraconazole at a dose of 100 mg once daily. At present, 2 yr after admission, the patient is in good clinical condition.

View larger version (18K): [in this window] [in a new window]   Figure 1.   The course of the galactomannan serum ratio (continuous line) during treatment with amphotericin B (solid box) and voriconazole (shaded box). Serum peak (dotted line) and through (dash line) levels of voriconazole and dose (mg/kg/dose) during intravenous (shaded box) and oral (open box) administration.

The presence of the Aspergillus antigen galactomannan was measured in the patient's serum with a commercial sandwich enzyme-linked immunosorbent assay (ELISA) (Plateliar R Aspergillus; Sanofi Diagnostics Pasteur, Marnes-La Coquette, France) (5). Briefly, 50 µl of a pretreated sample of 50 µl of horseradish peroxidase-conjugated EB-A2 monoclonal antibody (mAb), were placed in the wells of a microtiter plate coated with EB-A2 mAb and incubated at 37° C for 90 min. The plates were then washed, 100 µl of buffer containing ortho-phenylenediamine was added to the wells, and the plates were incubated for 30 min. The reaction was stopped with 50 µl 4M sulfuric acid, and the optical density (OD) was read at 492 nm. Negative serum spiked with 1 ng of Aspergillus antigen galactomannan per milliliter provided the threshold control, and a positive and negative control were included with each test. The serum ratio was calculated for each sample by dividing the OD of the sample by that of the threshold control, and ratios larger than 1.5 were considered positive, as recommended by the manufacturer.

The minimal inhibitory concentration (MIC) of itraconazole and voriconazole for the A. nidulans isolates was determined with an agar dilution method with RPMI 1640 medium (6). This assay has been shown to produce consistent and meaningful results for in vitro susceptibility testing of A. fumigatus with antifungal azoles. Briefly, doubling dilutions of itraconazole and voriconazole from 64 to 0.03 µg/ml were prepared, mixed with molten RPMI agar (Gibco, BRL, Life Technologies B.V., Breda, The Netherlands), and allowed to set. Spore suspensions of 10⁷ conidia/ml were made in phosphate-buffered saline (PBS), and the plates were inoculated with the suspensions, using a multipoint inoculator (Denley Multipoint Inoculator A400; Sussex, UK). The plates were incubated at 35° C and read after 48 h. An itraconazole-susceptible (MIC = 0.25 µg/ml) and an itraconazole-resistant (MIC > 64 µg/ml) A. fumigatus isolate were included in each test. The MIC was defined as the lowest concentration of drug at which there was no visible growth. The serum concentrations of voriconazole were measured with a bioassay that is recommended by the drug manufacturer.

The course of the Aspergillus antigen galactomannan titer in the serum during treatment is shown in Figure 1. The titer remained high during treatment with amphotericin B and initially during treatment with voriconazole. The titer increased after 1 mo of therapy, but started to decline 6 wk after therapy with voriconazole was begun. The titer continued to decline after switching to oral voriconazole therapy, and finally became negative 4 mo after the patient began voriconazole therapy. Galactomannan was not detected in serum samples obtained 7 and 8 mo after voriconazole therapy was begun, nor in samples obtained 3 mo after switching to itraconazole prophylaxis. The MIC for the cultured A. nidulans isolates was 0.25 µg/ml for itraconazole and 0.5 µg/ml for voriconazole. During treatment with voriconazole the peak levels in the serum varied between 2.7 and 6.0 µg/ ml, which was from 5.5- to 12-fold higher, respectively, than the MIC. The trough levels of voriconazole were between 1 and 5.5 times higher than the MIC during intravenous administration of the drug, but dropped below the MIC during oral therapy.

	DISCUSSION

TOP ABSTRACT INTRODUCTION DISCUSSION REFERENCES

Patients with CGD are highly susceptible to infection by catalase-positive microorganisms, including fungi such as Aspergillus species. The mortality of invasive aspergillosis in CGD is high despite specific antifungal treatment. Most infections are caused by A. fumigatus and A. flavus, but invasive infections caused by Aspergillus species with low pathogenicity, including A. niger, have been reported (7). Invasive infections caused by A. nidulans are uncommon, and most cases have been reported in patients with CGD, which suggests that these patients may be at increased risk for infections by this microorganism (7, 8). Thoracic-wall extension of pulmonary aspergillosis is rare and has been reported almost exclusively in patients with CGD (7, 9). Thoracic-wall invasion has been documented in patients infected with A. fumigatus and A. nidulans, and may present as osteomyelitis of the ribs or as cutaneous abscesses. Surgical drainage or resection of infected tissue is often required and may help to establish a diagnosis, since Aspergillus species can be readily cultured from tissue debris, as was the case in our patient.

The treatment of invasive aspergillosis in CGD is very difficult, especially in patients with absent or minimal oxidative metabolism by neutrophils, since the underlying immunodeficiency is the most important factor with respect to the outcome of treatment. Therefore, patients with CGD have been treated with either allogenic granulocyte transfusions from healthy donors (3) or with immunomodulative agents such as recombinant IFN- (1). The addition of these regimens to antifungal therapy with either amphotericin B or itraconazole has proved successful in some patients with CGD and established invasive aspergillosis (1, 2, 10). However, our patient did not respond to any of these treatment regimens, and also failed to benefit from local instillation of donor granulocytes (4). Clinical improvement was only observed after amphotericin B was discontinued and voriconazole was begun.

Voriconazole is a novel, broad-spectrum antifungal triazole that is fungicidal against a wide range of fungal organisms including Aspergillus species and other opportunistic molds (11). The drug can be administered both intravenously and orally, and only minor side effects have been reported. The in vitro MIC for voriconazole against A. nidulans was slightly higher than that of itraconazole, a situation that has also been reported for A. fumigatus. Nevertheless, voriconazole has been shown to have at least equivalent efficacy to that of itraconazole in increasing survival in a rat model of invasive infection with A. fumigatus, and voriconazole was better absorbed than itraconazole following oral administration (12). Although one clinical trial has shown good efficacy of voriconazole in patients with acute invasive aspergillosis (13), no data have yet been published for patients with invasive aspergillosis caused by non-fumigatus Aspergillus species. In our case voriconazole was very effective and well tolerated. The recommended dose for adults, 3 mg/kg twice daily, was initially used in our patient, but resulted in relatively low serum concentrations, which suggests that the drug is cleared more rapidly in children, as has been shown for more antifungal azoles such as fluconazole (14). In our patient the recommended peak serum levels of voriconazole of more than 3 µg/ml were achieved at a dose of 6 mg/kg twice daily. The galactomannan titer in the serum decreased during treatment, and may be useful for monitoring the response to antifungal therapy, although the decline was observed 6 wk after initiation of effective therapy.

Itraconazole has been shown to be effective for the treatment and prophylaxis of invasive aspergillosis in patients with CGD (10), but the drug has some well-known drawbacks, such as lack of an intravenous formulation and variable resorption from the intestine. In our case, the cultured A. nidulans isolate was susceptible in vitro to itraconazole, but therapy with this drug had to be discontinued because its administration by the oral route was no longer possible and an intravenous formulation was not available. Voriconazole may be an effective alternative agent for the treatment of invasive aspergillosis in patients with CGD, and therefore deserves further evaluation.