Pulmonary Cryptococcosis in Solid Organ Transplant Recipients: Clinical Relevance of Serum Cryptococcal Antigen

Abstract

Background

Role of serum cryptococcal antigen in the diagnosis and determinants of antigen positivity in solid organ transplant (SOT) recipients with pulmonary cryptococcosis has not been fully defined.

Methods

Study population included SOT recipients with pulmonary cryptococcosis in a prospective, multicenter study conducted between 1999 and 2006.

Results

Cryptococcal antigen was positive in 83% (40/48) of the patients with pulmonary cryptococcosis. Patients with concomitant extrapulmonary disease were more likely to have a positive antigen (p=0.018), and antigen titers were higher in those with extrapulmonary disease (p=0.003) or fungemia (p=0.045). Patients with single nodules were less likely to have a positive antigen than those with all other radiographic presentations (p=0.053). Among patients with isolated pulmonary cryptococcosis, lung transplant recipients were less likely to have positive cryptococcal antigen than other types of SOT recipients (p=0.003). In all, 38% of the patients were asymptomatic or had pulmonary cryptococcosis detected as an incidental finding. Nodular densities or mass lesions were more likely to present as asymptomatic or incidentally detected pulmonary cryptococcosis than pleural effusions and infiltrates (p=0.008).

Conclusions

A positive serum cryptococcal antigen in SOT recipients with pulmonary cryptococcosis appears to reflect extrapulmonary or more advanced radiographic disease.

Introduction

Cryptococcosis occurs in 0.3-5.3%, and in an average of 2.8% of solid organ transplant (SOT) recipients (1-3) Approximately 25-54% of transplant recipients with cryptococcosis have pulmonary disease and in 6-33% disease is limited to the lungs (4, 5). Disseminated infection, including central nervous system (CNS) disease and fungemia occur in 52-61% and 20-25% of SOT recipients, respectively (2, 6, 7).

Cryptococcal antigen assays that measures polysaccharide released from C. neoformans has proven to be a valuable diagnostic assay for cryptococcosis (8-10). The sensitivity and specificity of serum antigen for the diagnosis of cryptococcal meningoencephalitis largely approaches that of the cerebrospinal fluid (CSF) (11, 12). The test however, is considered to be of limited value for the diagnosis of pulmonary cryptococcosis (13-17). Case series have documented a wide variability in the frequency of positive serum antigen, ranging from 33-100% in SOT recipients with pulmonary cryptococcosis (2, 5, 18-21). The diagnostic utility and determinants of serum cryptococcal antigen positivity in SOT recipients with pulmonary cryptococcosis has not been fully defined. A prospective database comprising a large cohort of organ transplant recipients with cryptococcosis offered a unique opportunity to address these issues (6). The goals of this study therefore, were to determine the role of serum cryptococcal antigen in the diagnosis of pulmonary cryptococcosis, variables influencing antigen positivity, and its prognostic implications in SOT patients with pulmonary cryptococcosis.

Methods

Study population was derived from a cohort of organ transplant recipients with cryptococcosis in a prospectively conducted multicenter study between 1999 and 2006. Detailed description of this cohort has been reported elsewhere (6). None of the patients were HIV-infected. Cryptococcal disease was defined as per the criteria proposed by the European Organization for Research and Treatment in Cancer and the Mycosis Study Group (22). Of 122 SOT recipients with cryptococcosis, 66 had pulmonary cryptococcal disease. Pulmonary lesion was considered to be due to cryptocccosis if the yeast was documented in a histopathologic specimen from the lung, or a respiratory tract or pleural fluid culture yielded C.neoformans. Pulmonary lesion in patients with positive serum cryptococcal antigen, fungemia, or positive culture for C.neoformans from the CSF was also considered to be due to cryptococcosis if an alternative etiology for the pulmonary lesion could not be established and resolution was documented with antifungal therapy for cryptococcosis. Organ sites involved were classified as CNS; pulmonary; skin, soft-tissue, and osteoarticular; or other (2, 6). Disseminated infection was defined as CNS infection or fungemia or involvement of >2 noncontiguous organ sites (6). The mortality rate was assessed at 90 days (6).

Patients with abnormal imaging findings on chest radiographs were evaluated with computerized tomographic studies and location, and radiologic characteristics of the lesions were recorded. Interpretation of the radiological studies was per the radiologists at each center. When possible, the reports of radiologic studies were enclosed with the data collection form. Cryptococcal antigen testing was performed as part of standard clinical care at that institution. For the purpose of this study, antigen titer at baseline i.e. at the time of diagnosis of pulmonary cryptococcosis and before employment of antifungal therapy was considered. The results of antigen testing were available in 48 of 66 patients with pulmonary cryptococcosis.

Statistical analyses were performed using Intercooled Stata version 9.2 (College Station, TX). Categorical data were compared using the Chi-square test. The Fisher’s exact test was used for contingency tables with small numbers. Antigen titers were log transformed and compared using the Wilcoxon rank sum test. The time of onset of disease post transplant was also evaluated using the Wilcoxon rank sum test.

Results

The diagnosis of pulmonary cryptococcosis was based on a positive respiratory tract or pleural fluid culture in 39.4% (26/66), needle or excision biopsy of the pulmonary lesion in 30.3% (20/66), positive serum antigen in 12.1% (8/66), positive CSF culture in 12.1% (8/66), fungemia in 4.5%(3/66), and on autopsy in 1.5% (1/66) of the patients. Clinical and demographic characteristics of 48 patients for whom the results of antigen testing were available are outlined in Table 1. A positive antigen was documented in 83.3% (40/48) of the patients (median titer 1:64). When cases diagnosed on the basis of a positive antigen were excluded (n=8), 80% (32/40) of the patients with pulmonary cryptococcosis had a positive serum cryptococcal antigen.

Table 1.

Clinical and demographic characteristics of the patients with pulmonary cryptococcosis

Number	48
Age, median (range)	53.2(22-77)
Gender, %male	75% (36/48)
Type of transplant
Kidney	33.3%(16/48)
Liver	27.1%(13/48)
Heart	14.6%(7/48)
Kidney-pancreas	10.4%(5/48)
Lung	8.3%(4/48)
Multiorgan	6.2%(3/48)
Immunosuppressive agents
Tacrolimus	89.0%(43/48)
Cyclosporine A	8.3%(4/48)
Azathioprine/mycophenolate mofetil	1%(1/48)
Prednisone	75%(36/48)
Median dose	7.5 mg
Time to onset of cryptococcosis
Median (interquartile range)	16 months (7.7-26 months)
Extrapulmonary involvement*
Central nervous system	31.2%(15/48)
Fungemia	22.2%(10/48)
Other sites**	6%(3/48)
Radiographic presentation
Multiple nodules	10
Infiltrate (alveolar or nodular)	10
Simple nodule	8
Consolidation	8
Mass	4
Cavitary	3
Pleural effusion	3
Not specified	2

^*May have more than one site of involvement.

^**These included cutaneous and bone involvement 1, peritoneal fluid 1, and prostatitis 1.

Eighteen (37.5%) of the 48 patients with pulmonary cryptococcosis disease also had extrapulmonary cryptococcal disease (Table 1). Patients with concomitant extrapulmonary disease were more likely to have a positive serum antigen (100%, 18/18 vs. 73.3%, 22/30 vs., p=0.018) and had higher antigen titer (1:512 vs.1:32 vs., p=.003) than patients who had cryptococcosis limited to the lungs. The rates of antigen positivity did not differ significantly for patients with fungemia compared to those without fungemia (90% vs. 80%, p=0.66) however, antigen titer was significantly higher in fungemic than non-fungemic patients with pulmonary cryptococcosis (1:512 vs. 1:32, p=.045). Overall, 47.5% (19/40) of the patients with positive antigen had extrapulmonary disease compared to 12.5% (1/8) with negative antigen (p=0.07).

Most common radiographic presentation of pulmonary cryptococcosis was nodules (38%, 18/48) that were multiple in 10 and single in 8 patients (Table 1). Serum antigen was less likely to be positive in patients with single nodule compared to those with all other radiographic presentations (62.5%, 5/8 vs. 89%, 34/38, p=.053).

Pulmonary cryptococcosis limited to the lungs

In 63% (30/48) of the patients, cryptococcal disease was limited to the lungs. Of these, 73% (22/30) had positive cryptococcal antigen. Cases with positive antigen occurred 16.9 months post-transplant and those with negative antigen 33.7 months post-transplant ( p=0.19, Table 2). A positive antigen was documented in 91% of the kidney, 83% of the liver, 80% of the heart, and none of the lung transplant recipients (p=0.01) (Table 2). Fifty percent (4/8) of the patients with negative cryptococcal antigen were lung transplant recipients compared to 0% (0/22) of those with positive antigen (p=0.003). Cryptococcal antigen was positive in 25% of the patients with single nodule and 87.5% of those with multiple nodules (Table 2). Other variables outlined in Table 2 were not significantly different for patients with positive versus negative antigen.

Table 2.

Variables associated with serum cryptococcal antigen positivity in patients with pulmonary cryptococcosis limited to the lungs (n=30).

Variable	Patients with positive antigen (n=22)	Patients with negative antigen (n=8)	P-value*
Type of transplant			0.01
Kidney	46%(10/22)	13%(1/8)
Liver	23% (5/22)	13%(1/8)
Kidney-pancreas	14%(3/22)	13%(1/8)
Heart	18%(4/22)	13%(1/8)
Lung		50%(4/8)
Time to onset, median	16.9 months	33.7 months	0.19
Immunosuppressives			0.99
Tacrolimus	86%(19/22)	88%(7/8)
Cyclosporine A	14%(3/22)	13%(1/8)
Prednisone	73%(16/22)	88%(7/8)
Radiographic characteristic			0.21
Multiple nodules	32%(7/22)	13%(1/8)
Single nodule**	5%(1/22)	37.5%(3/8)
Infiltrate (nodular)	5%(1/22)	0
Infiltrate (alveolar)	9%(2/22)	25.0%(2/8)
Cavitary	9%(2/22)	13%(1/8)
Consolidation	23%(5/22)	13%(1/8)
Mass	14%(3/22)	0
Pleural effusion	0	0
Mortality	5%(1/22)	0/8	0.99

^*The p-values indicate the overall difference between variables in the 2 groups.

^**Single nodule versus other radiographic presentations (p=0.05)

Symptomatic versus asymptomatic or incidentally detected pulmonary cryptococcosis

Overall, 62.1% (41/66) of the patients with pulmonary cryptococcosis had symptomatic disease and 37.9% (25/66) had either no pulmonary symptoms or had pulmonary cryptococcosis detected as an incidental finding. Median time to onset was 21 months post-transplant for asymptomatic or incidental lesions and 11.6 months for symptomatic pulmonary lesions (p=0.10) (Table 3). Type of organ transplant or primary immunosuppressive regimen did not correlate with symptomatic versus asymptomatic lesions (Table 3). However, prednisone dose (within 6 months of the diagnosis of cryptococcal disease) was significantly higher for patients who were symptomatic compared to those who were asymptomatic or had incidental pulmonary lesions (p=0.005, Table 3).

Table 3.

Demographic and clinical variables in patient with symptomatic versus asymptomatic or incidentally detected pulmonary cryptococcosis.

Variable	Asymptomatic/incidental cases (n=25)	Symptomatic cases (n=41)	P-value
Type of transplant			0.56
Kidney	36%(9/25)	34%(14/41)
Liver	24%(6/25)	29%(12/41)
Lung	8%(2/25)	10%(4/41)
Heart	20%(5/25)	10%(4/41)
Kidney-pancreas	12%(3/25)	7%(3/41)
Multiorgan	0/25	10%(41)
Immunosuppression			0.80
Tacrolimus	84%(21/25)	78%(32/41)
Cyclosporine A	12%(3/25	15%(6/41)
Azathioprine/mycophenolate  mofetil	4%(1/25)	7%(3/41)
Prednisone,median dose(mg)	6	10	0.005
Time to onset, median	21 months	11.6 months	0.10
No. with positive antigen¶	89%(16/18)	80%(24/30)	0.69
titer	1:32	1:128	0.14
Radiographic characteristic			0.06
Consolidation	24%(6/25)	18%(7/38)*
Single nodule	24%(6/25)	18%(7/38)
Multiple nodule	32%(8/25)	13%(5/38)
Mass	8%(2/25)	5%(2/38)
Cavitary	8%(2/25)	5%(2/38)
Infiltrate (alveolar)**	0/25	18%(7/38)
Pleural effusion**	0/25	8%(3/38)
Infiltrate (Nodular)**	4%(1/25)	11%(4/38)

^¶Data are presented for 48 patients for whom the results of serum cryptococcal antigen were available.

^*Data are presented for 38 patients for whom radiologic characteristics of the pulmonary lesion were available.

^*Pleural effusion or infiltrates compared to all other types of lesions were more likely to present as symptomatic disease (p=0.008).

Asymptomatic or incidentally detected pulmonary cryptococcosis presented most frequently as multiple nodules (32%), single nodules (26%), or consolidation (24%)(Table 3). Patients with pleural effusions or infiltrates were significantly more likely to have symptomatic as opposed to asymptomatic or incidentally detected lesions (37% vs. 4%, p= .002).The antigen titer was significantly higher in symptomatic than in asymptomatic or incidentally diagnosed cases (1:164 vs. 1:32, p=.02).

Outcome

Mortality at 90 days did not correlate with serum antigen positivity; 10% (4/40) of the patients with positive antigen and 0% (0/8) of those with negative antigen died (p=0.99). Median antigen titer in patients who died was 1:256 compared to 1:64 in those who lived (p= 0.78). Mortality rate also did not correlate with antigen positivity in patients with isolated pulmonary cryptococcosis (5%, 1/22 versus 0%, 0/8, p=0.99). However, patients with symptomatic compared to those with asymptomatic or incidentally detected disease had a higher mortality rate (24%, 10/41 versus 4%, 1/25, p=0.04).

Discussion

Several observations from our study are clinically relevant with regards to pulmonary cryptococcosis in SOT recipients. A positive cryptococcal antigen in these patients appears to reflect extrapulmonary disease. Cryptococcal infection is acquired by inhalation of the basidiospores from the environment with subsequent contaminant of the yeast by granulomatous inflammatory responses (23). Establishment of latency is proposed to occur in a primary complex consisting of pulmonary lesion and hilar lymph nodes (24-26). A vast majority of cryptococcal disease is considered to be due to reactivation of quiescent or latent infection (27, 28). Fungemia and CNS infection result from lymphohematogenous dissemination from of an overt or subclinical pulmonary focus.

Granuloma formation signifies an effective host response against cryptococcus and well circumscribed pulmonary nodules represent walled off or quiescent granulomas (24). Disrupted granulomatous lesions with spread to surrounding lung parenchyma represent more advanced stages of infection and reflect inability of the host to contain the yeast. For example, in severely immunocompromised patients such as those with HIV infection, pulmonary cryptococcosis is more likely to present as interstitial infiltrates and alveolar lesions as opposed to nodular densities (13, 24, 29, 30). Additionally, those with higher CD4+ counts are more likely to have localized pulmonary lesions (31).

These pathophysiologic considerations yield important insights into the evolution of pulmonary cryptococcosis and provide a rational explanation for several findings in our study. Rates of antigen positivity and titer correlated with evidence of disseminated disease and/or fungemia. A major determinant of antigen positivity was also the extent and characteristics of the pulmonary lesion. Patients with single nodules were less likely to have a positive antigen than those with more advanced lesions radiographically.

Approximately, one-third of the SOT recipients with pulmonary cryptococcosis in our study had asymptomatic or incidentally detected pulmonary lesions. Patients with nodular densities or mass lesions were less likely to be symptomatic than those with pleural effusions and infiltrates. A significant correlate of symptomatic disease was also the prednisone dose at diagnosis. Receipt of corticosteroids is a well recognized risk factor for cryptococcosis (20, 32). Although the daily dose of prednisone that confers an increased risk is not known, dosages ≥ 10 mg per day in non-transplant setting have been shown to place the patients at a higher risk for cryptococcal disease (33).

In patients with isolated pulmonary cryptococcosis, antigen positivity was lower for lung compared to all other types of organ transplant recipients. A positive serum cryptococcal antigen is suggestive of a high burden of organisms and the high titers often correlate with advanced or more severe disease (34). That lung transplant recipients who are typically more immunosuppressed were less likely to have positive cryptococcal antigen therefore appears intuitively paradoxical. However, in a prior report on pulmonary cryptococcosis in non-HIV patients, serum cryptococcal antigen was also negative in lung transplant recipients (18). It is possible that more frequent and closer follow-up with pulmonary imaging studies may have allowed earlier detection of pulmonary cryptococcosis in lung transplant recipients. For example, in many lung transplant programs, chest radiographs are routinely performed at monthly and at even more frequently in the early posttransplant period (35). However, previous studies have shown that lung transplant recipients even when controlled for the immunosuppressive regimen had a lower risk of disseminated cryptococcosis (6), and despite a higher incidence of Aspergillus infections, lung transplant recipients also have a lower risk of disseminated invasive aspergillosis (36, 37). Thus it is likely that factors other than closer scrutiny such as unique host immunological defects or other yet poorly understood variables play a role in determining the risk of disseminated disease in various types of SOT recipients. We however note that our findings should be interpreted with caution given a small number of lung transplant recipients in the study.

Several limitations of this study deserve to be acknowledged. Cryptococcal antigen assay was performed locally thus leading to the possibility of interlaboratory variability in test results. However, in over 40 years since the development of this test, the technology has become standardized and it is widely used in clinical practice. Second, although standard terminology was utilized for the description of the imaging findings (38, 39), radiographic studies were interpreted by radiologists at each center. Finally, our study was unable to determine the role of serum cryptococcal antigen monitoring in patients with pulmonary cryptococcosis. High serum cryptococcal antigen titer was a prognostic factor for poor outcomes (40) and persistently high serum cryptococcal antigen titers correlated significantly with the risk of relapse in HIV-infected patients with cryptococcosis(41). In other studies, antigen monitoring was shown to be of limited value in the management of cryptococcal disease (8, 42, 43).

In summary, our study shows that antigen positivity in SOT recipients with pulmonary cryptococcosis appears to reflect extrapulmonary involvement or more advanced radiographic disease. These data provide a rational explanation for variability in the rates of antigen positivity in the literature in SOT recipients with pulmonary cryptococcosis and have implications relevant for the interpretation of cryptococcal antigen results in these patients.