Methods

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Patients

Between 1990 and 1994, 56 patients received lung transplants at the University Hospital Groningen, the Netherlands; these patients had a 1-year survival rate of 84%. We reviewed the data of four male patients, 33 to 58 years of age, who had received lung transplants in 1993 and had had at least one episode of proven A. fumigatus infection. The indications for lung transplantation were emphysema in two patients and cystic fibrosis in two patients. The diagnosis of invasive fungal infection required isolation of fungus from a normally sterile site or evidence of fungal tissue invasion in a biopsy specimen. Key clinical and laboratory findings of the patients are summarized in Table 1.

Serum Specimens

Serum samples from all four patients were collected after lung transplantation and stored at – 20 °C. A positive reference serum sample was prepared by pooling the serum specimens of several patients with diseases related to A. fumigatus. These patients (who had pulmonary aspergilloma or allergic bronchopulmonary aspergillosis) showed high IgG antibody titers specific for A. fumigatus on enzyme-linked immunosorbent assay (ELISA) and had high numbers of precipitins on double immunodiffusion tests.

Immunochemical Procedures

We measured the IgG antibody response specific for A. fumigatus antigens (A. fumigatus IgG) retrospectively and in batches by a direct ELISA procedure [8] with late-phase Sabouraud dextrose culture filtrates as previously described [9]. The results are expressed as ELISA units as previously described [8].

Results

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The A. fumigatus IgG levels for patient 1 during disease progression and treatment are shown in Figure 1. Before lung transplantation, this patient's A. fumigatus IgG levels were low (data not shown). After lung transplantation, these levels remained low for 246 days. Within 21 days after this period (246 to 267 days after transplantation), the levels increased sharply, approximately 1 to 2 weeks before cavitations were seen in the left lung (day 275 after transplantation) and in the right lung (day 281 after transplantation) (Figure 1). In the following period, A. fumigatus IgG levels remained high and even increased slightly. When aspergilloma could be diagnosed by transthoracic puncture on day 408 after transplantation, the levels were still elevated (Figure 1). During antifungal treatment, the levels steadily decreased until day 587 after transplantation, although sputum samples repeatedly grew A. fumigatus. When fungal reinfection was diagnosed by transthoracic puncture on day 688 after transplantation, the A. fumigatus IgG level had sharply increased.

View larger version (18K): [in this window] [in a new window]   Figure 1. Aspergillus fumigatus IgG readings from enzyme-linked immunosorbent assay (ELISA) of patient 1 plotted against time. Arrow 1 indicates day of cavitation of left upper lobe, arrow 2 indicates day of cavitation of right upper lobe, arrow 3 indicates day of diagnosis of aspergilloma of the left upper lobe, and arrow 4 indicates day of diagnosis of fungal reinfection of the left upper lobe. The dashed line represents an interpolation of the A. fumigatus IgG response; no serum specimens were available during this period.

Data from the four patients are summarized in Table 1. Increasing A. fumigatus IgG levels closely paralleled cytologic or microbiological identification of A. fumigatus from bronchoalveolar lavage fluid (patient 3), deterioration of lung function, the development of an infiltrate on the radiograph, clinical symptoms, and the diagnosis of organizing pneumonia with A. fumigatus (patient 2).

Increasing A. fumigatus IgG levels were found to precede radiographic identification of lung cavitations (by 1.5 weeks in patient 3 and by 1 to 2 weeks in patient 1), the diagnosis of aspergilloma by radiologic and cytologic examination of bronchoalveolar lavage fluid (by 1.5 weeks in patient 3) or by cytologic examination of material obtained by transthoracic puncture (by 20 weeks in patient 1), and the diagnosis of thoracotomy wound infection (by 2 weeks in patient 4) (Table 1). In patient 1, highly elevated A. fumigatus IgG levels were found at the time of fungal reinfection and may have increased before reinfection was diagnosed (Figure 1). However, because no serum specimens were available between 587 and 688 days after transplantation, we could not test this assumption. The rate of increase of A. fumigatus IgG levels was remarkable; titers changed within 2 weeks from negative to strongly positive values in all patients studied (data not shown).

Successful antifungal treatment was reflected by clinical improvement in all patients and by decreasing A. fumigatus IgG levels in three patients (patients 1, 2, and 3). Decreasing A. fumigatus IgG levels correlated with the inability to culture or identify A. fumigatus from bronchoalveolar lavage fluid (patients 1 and 3) and with radiographic improvement (patients 1 and 2). In all patients, A. fumigatus IgG levels peaked 2 to 3 weeks after initiation of liposomal amphotericin B treatment and again after itraconazole treatment Figure 1, as previously described; this outcome may have reflected the liberation of antigen after the killing of the microorganism [10].

Discussion

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We present data from four retrospectively studied patients, who had proven A. fumigatus infections after lung transplantation and fatal outcome. These data show a close correlation between A. fumigatus IgG levels measured in serum and radiographic features, cytologic and microbiological findings, and the clinical diagnosis of fungal disease.

Two of the four patients had a clear diagnosis of aspergilloma formation either in one of the transplanted lungs (patient 1) or in the native lung (patient 3). One patient (patient 2) presented with organizing pneumonia with A. fumigatus, and another patient (patient 4) developed A. fumigatus infection of the thoracotomy wound. All patients were treated systemically with itraconazole, liposomal amphotericin B, or both; they improved considerably with treatment. Antifungal treatment of the fungal reinfection in patient 1 was not successful. The increase in A. fumigatus IgG levels in patient 2 that occurred 222 days after transplantation may have indicated reinfection with A. fumigatus at a subclinical level. However, no evidence of reinfection was found at autopsy. In patient 1, however, reinfection with A. fumigatus was reflected by an increased A. fumigatus IgG response and was confirmed by transthoracic puncture and autopsy. In patient 4, A. fumigatus IgG levels did not decrease after liposomal amphotericin B treatment was stopped until 243 days after transplantation (data not shown).

After lung transplantation, patients are at high risk for infections of the lung and airways [5]. This is generally attributed to a decrease in both the humoral and the cellular immune defenses, which is caused by immunosuppressive therapy. This assumption was supported by recent in vitro studies [11] that showed decreased IgG formation by B cells in the presence of alveolar macrophages in patients after lung transplantation.

In contrast to these in vitro studies, we found A. fumigatus IgG levels to be as high as or higher than the levels found in serum specimens from patients with aspergilloma (without lung transplantation) or exacerbation of allergic bronchopulmonary aspergillosis. Additionally, dramatic changes in A. fumigatus IgG levels from negative to strongly positive values within 2 to 3 weeks indicate that the immune suppression therapy used to prevent rejection does not impair the humoral immune response to invading fungi. Thus, the high susceptibility to fungal infection of this patient group is not explained by a diminished humoral immune response. Our observations are similar to those seen in patients with cytomegaloviral infections after lung transplantation, in whom high antibody responses in serum can be detected during infection episodes [12].

Fungal infections are a major concern; those caused by A. fumigatus are especially serious given the high mortality rate caused by this microorganism [13]. In addition to being a direct cause of death, frequent infection may lead to long-term sequelae associated with increased episodes of rejection and possibly to the development of obliterative bronchiolitis. An association of pulmonary bacterial infections [14] and particularly viral infections [15] with obliterative bronchiolitis has been described.

The diagnosis of fungal infection is difficult and often is not established before death [16]. A slight increase in body temperature and new infiltrations on chest radiography are not specific for fungal infection. Chest radiographs [17] and spirometric findings [18] are abnormal in both rejection and infection of the lungs. Besides, patients may have pulmonary infections with A. fumigatus even though chest radiography shows a normal appearance [19]. Recent studies [5] have shown that invasive A. fumigatus can be found during the autopsies of patients not suspected of having invasive disease.

Patients are susceptible to lung rejection and infection after lung transplantation, two recurrent problems that require early and accurate diagnosis and treatment. Transbronchial biopsy improves the accuracy of diagnosis of acute lung rejection and often allows the distinction to be made between rejection and infection [20]. Our study, although limited in size, suggests that elevated A. fumigatus IgG serum levels in lung allograft recipients may provide supplemental information helpful in diagnosing and treating pulmonary Aspergillus infections and in monitoring the success of antifungal treatment. The sensitivity and specificity of A. fumigatus IgG serologic testing in a larger group of patients who have had lung transplantation are currently being studied.

Dr. Mannes: Department of Pulmonology, Leyenburg Hospital, Leyweg 275, 2545 CH The Hague, the Netherlands.

Drs. van der Bij, van der Werf, and Koeter: Department of Pulmonology, Clinic for Internal Medicine, University Hospital Groningen, Hanzeplein 1, 9713 gZ Groningen, the Netherlands.

Dr. de Boer: Department of Cardiothoracic Surgery, University Hospital Groningen, Hanzeplein 1, 9713 gZ Groningen, the Netherlands.