Extent of Lung Involvement and Serum Cryptococcal Antigen Test in Non-Human Immunodeficiency Virus Adult Patients with Pulmonary Cryptococcosis.

BACKGROUND: Serum cryptococcal antigen (CrAg) test is the most used noninvasive method to detect cryptococcal infection. However, false-negative CrAg test is not uncommon in clinical practice. Then, the aim of this study was to investigate the factors associated with false-negative CrAg test among non-human immunodeficiency virus (HIV) adult patients with pulmonary cryptococcosis and its clinical features.
METHODS: One hundred and fourteen non-HIV adult patients with pulmonary cryptococcosis, proven by biopsy, were retrospectively reviewed. Finally, 85 patients were enrolled; 56 were CrAg positive (CrAg+ group) and 29 were negative (CrAg- group). It was a cross-sectional study. Then, baseline characteristics, underlying diseases, clinical symptoms, laboratory findings, and chest radiological findings were reviewed and analyzed. Chi-square test was used to analyze categorical variable. Odds ratio (OR) was used to measure correlation. Student's t- test was obtained to analyze continuous variable.
RESULTS: No difference in baseline characteristics, underlying diseases, clinical symptoms, and laboratory findings were found between two groups (P > 0.05 in all). Nevertheless, diffuse extent lesion was 82.1% in CrAg+ group and 10.3% in CrAg- group (χ2 = 40.34, P < 0.001; OR = 39.87).
CONCLUSIONS: Among patients with limited pulmonary involvement, a negative serum CrAg does not preclude the diagnosis of pulmonary cryptococcosis. However, among patients with extensive pulmonary involvement, serum CrAg is a useful diagnostic tool for pulmonary cryptococcosis. Furthermore, we also noticed that the untypical and mild presentations with extensive pulmonary lesion might be the features of pulmonary cryptococcosis, which needs further investigation.

INTRODUCTION

Cryptococcus neoformans-Cryptococcus gattii species complex is a nonmycelial, budding encapsulated yeast-like fungus, which can lead to significant infections, ranging from asymptomatic pulmonary colonization to lethal meningoencephalitis.[12] Although cryptococcosis is a typical systemic opportunistic pathogen in immunocompromised hosts such as human immunodeficiency virus (HIV) infection, pulmonary cryptococcosis is not rare in patients without recognized immunologic defect.[34] Some studies showed that the most common radiological presentations of pulmonary cryptococcosis were lung nodules and masses that required differentiation from lung cancer.[56] Several studies demonstrated that symptoms of pulmonary cryptococcosis are often untypical and can emulate those of lung cancer including cough and expectoration.[78] Biopsy of suspected pulmonary cryptococcosis from lung nodule or mass can be challenging. At present, serum cryptococcal antigen (CrAg) test is the most used noninvasive method to detect cryptococcal infection. However, in clinical practices, having negative serum CrAg is common among patients with pulmonary cryptococcosis. The purpose of the current study was to explore the factors associated with positive serum CrAg test among non-HIV adult patients with biopsy-proven pulmonary cryptococcosis.

METHODS

Ethical approval

This cross-sectional study was approved by the Research Ethics Committees of the Zhujiang Hospital of Southern Medical University (No. 2016-HXNK-007) and was conducted according to the Declaration of Helsinki. The clinical information, records, and data involved in this study were analyzed anonymously without intervention. Thus, informed consent was waived.

Study subjects

From January 2012 to December 2016, HIV-negative adult patients (more than 18 years old) with pulmonary cryptococcosis from Zhujiang Hospital of Southern Medical University, Second Affiliated Hospital of Chongqing Medical University, and First Affiliated Hospital of Chengdu Medical College were enrolled in our study. Pulmonary cryptococcosis was diagnosed by percutaneous transthoracic needle biopsy (PTNB) or postoperative biopsy with hematoxylin and eosin, periodic acid–Schiff, and methenamine silver staining. Meanwhile, the patients combined with extrapulmonary cryptococcosis, such as cerebral cryptococcosis, were excluded from our investigation. According to the criteria, 114 non-HIV adult patients with pulmonary cryptococcosis, proven by biopsy, were retrospectively reviewed. Finally, 85 patients were enrolled; 56 were CrAg positive (CrAg+ group) and 29 were negative (CrAg− group) [Figure 1].

Figure 1

Flow diagram of the study patient selection. Two HIV-positive patients combined with extrapulmonary cryptococcosis. Two HIV-positive patients combined with pulmonary cryptococcosis. One pulmonary cryptococcosis patient was under 18 years old. HIV: Human immunodeficiency virus.

Data and information collection

The data and information were collected without intervention, including age, gender, symptoms, chest radiological findings, underlying diseases, concentrations of hemoglobin (Hb), platelet counts (PLT), white blood cell (WBC) counts, neutrophils counts, and plasma procalcitonin (PCT) level. Then, serum CrAg was detected by lateral flow assay (IMMY, Norman, OK, USA). Symptom scores, the number of symptoms a patient presented, were calculated. Chest radiological findings were reviewed. We divided the radiological findings into two conditions, diffuse extent lesion and limited extent lesion. Then, we defined diffuse extent lesion as lesions in multiple lobes, multiple lesions in a single lobe, or the lesion diameter >3 cm in a single lobe. The limited extent lesion was defined as the lesion (the diameter <3 cm) in a single lobe.

Data analysis

Statistical analyses were performed with SPSS software, version 17.0 (SPSS Inc., Chicago, IL, USA). Chi-square test was used to analyze categorical variable. Odds ratio (OR) was used to measure correlation. Student's t-test (mean ± standard deviation) was obtained to analyze continuous variable. P < 0.05 indicated a significant difference.

RESULTS

Patients' characteristics

One hundred and fourteen patients were diagnosed with pulmonary cryptococcosis by PTNB or postoperative biopsy between January 2012 and December 2016 from Zhujiang Hospital of Southern Medical University, Second Affiliated Hospital of Chongqing Medical University, and First Affiliated Hospital of Chengdu Medical College. Moreover, 29 patients were excluded by excluding criteria. Finally, 50 males and 35 females were enrolled in our study. The serum CrAg test was positive in 56 participants and was negative in 29 participants [Figure 1 and Table 1].

Table 1

Baseline characteristics of non-HIV adult patients with pulmonary cryptococcosis

Characteristics	CrAg+	CrAg−	Statistical values	P
n	56	29
Male gender, n (%)	34 (60.7)	16 (55.2)	0.242*	0.623
Age (years)	45.8 ± 10.0	49.9 ± 12.5	−1.646†	0.103
Weight (kg)	64.84 ± 14.00	62.37 ± 11.10	0.821†	0.414
Symptoms
Cough, n (%)	44 (78.6)	18 (62.1)	2.636*	0.104
Expectoration, n (%)	31 (55.4)	13 (44.8)	0.848*	0.357
Chest tightness, n (%)	11 (19.6)	6 (20.7)	0.013*	0.909
Hemoptysis, n (%)	5 (8.9)	1 (3.4)	0.875*	0.350
The numbers of symptoms (symptom scores)	1.52 ± 0.95	1.31 ± 0.98	0.947†	0.347
Underlying diseases, n (%)	10 (17.9)	5 (17.2)	0.005*	0.944
T2DM, n	4	2
Kidney transplantation, n	2	2
SLE, n	1	0
Astrocytoma, n	1	0
Nephrotic syndrome, n	1	0
Nasopharyngeal cancer, n	1	1
Langerhans cell histiocytosis, n	0	1
Hb (g/L)	132.85 ± 18.27	133.24 ± 17.07	−0.094†	0.925
Platelet (109/L)	266.98 ± 59.28	262.86 ± 76.13	−1.060†	0.292
WBC (109/L)	6.98 ± 1.69	7.04 ± 2.42	−0.133†	0.895
Neutrophils (109/L)	4.82 ± 1.40	4.89 ± 1.83	−0.171†	0.865
Plasma procalcitonin level (ng/ml)	0.11 ± 0.16	0.14 ± 0.24	−0.799†	0.427

*Chi-square test; †t-test. T2DM: Type 2 diabetes mellitus; SLE: Systemic lupus erythematosus; Hb: Hemoglobin; HIV: Human immunodeficiency virus; CrAg: Cryptococcal antigen; WBC: White blood cell.

Underlying diseases

Fifteen cases (17.6%) complicated with underlying diseases. Among them, type 2 diabetes mellitus (T2DM) (7.1%) and kidney transplantation (4.7%) were the two most common underlying diseases. Moreover, one case was diagnosed as kidney transplantation combined with T2DM. Then, other underlying diseases included systemic lupus erythematosus (1.2%), astrocytoma (1.2%), nephrotic syndrome (1.2%), nasopharyngeal cancer (2.4%), and Langerhans cell histiocytosis (1.2%). Then, no significant difference in rates of underlying diseases was observed between two groups.

Clinical symptoms

As shown in Table 1, cough (72.9%) and expectoration (51.8%) were the most common symptoms in patients with pulmonary cryptococcosis. Other common symptoms included chest tightness (20.0%) and hemoptysis (7.1%). Then, no significant difference in types of symptoms and symptom scores (1.52 ± 0.95 vs. 1.31 ± 0.98, P = 0.944) was observed between two groups.

Laboratory findings

There was no significant difference in Hb (132.85 ± 18.27 vs. 133.24 ± 17.07, P = 0.925), PLT (266.98 ± 59.28 vs. 262.86 ± 76.13, P = 0.292), WBC (6.98 ± 1.69 vs. 7.04 ± 2.42, P = 0.895), neutrophils (4.82 ± 1.40 vs. 4.89 ± 1.83, P = 0.865), and plasma PCT level (0.11 ± 0.16 vs. 0.14 ± 0.24, P = 0.427) between two groups [Table 1].

Chest radiological findings

Table 2 figured out that diffuse extent lesion was 82.1% in CrAg+ group and 10.3% in CrAg− group (χ2 = 40.34, P < 0.001). Moreover, OR was 39.87. Chest radiological findings and biopsy results in patients with pulmonary cryptococcosis were shown in Figure 2.

Table 2

Chest radiological findings of non-HIV adult patients with pulmonary cryptococcosis

Chest radiological characteristics	CrAg+ (n = 56)	CrAg− (n = 29)	χ2	P
Diffuse extent lesion, n (%)	46 (82.1)	3 (10.3)	40.34	<0.001
Limited extent lesion, n (%)	10 (17.9)	26 (89.7)

HIV: Human immunodeficiency virus; CrAg: Cryptococcal antigen.

Figure 2

The chest radiological findings and biopsy results in patients with pulmonary cryptococcosis. The figures were obtained from three different individuals ([a and d] from case 1, [b and e] from case 2, and [c and f] from case 3). Cases 1 and 2 from CrAg+ group were diagnosed by PTNB and Case 3 from CrAg− group was diagnosed by postoperative biopsy (frozen biopsy). Chest radiological findings (a-c): (a) Multiple lesions in a single lobe and the lesion diameter >3 cm in a single lobe (diffuse extent lesion); (b) lesions in multiple lobes (diffuse extent lesion); (c) lesion (the diameter <3 cm) in a single lobe (limited extent lesion). Biopsy results (d-f): The figures (H and E staining) demonstrates a representative view (×400). Lesion (orange arrows); Cryptococcus sp. yeasts (blue arrows). PTNB: Percutaneous transthoracic needle biopsy; H and E: Hematoxylin and eosin.

DISCUSSION

In the present study, our data showed that, among patients with suspected pulmonary cryptococcosis, in context of extensive pulmonary involvement, a negative serum CrAg is highly suggestive of an alternate diagnosis. In addition, among patients with limited pulmonary involvement, a negative serum CrAg does not preclude the diagnosis of pulmonary cryptococcosis. Furthermore, our results also suggest that, among patients with the extensive pulmonary lesion, serum CrAg is a useful diagnostic tool for pulmonary cryptococcosis.

Cryptococcosis has been considered a disease of opportunistic infection, mainly, in immunocompromised patients with HIV/AIDS, cancers, and immunosuppressive treatment.[910] Moreover, C. neoformans was the predominant etiological agent of cryptococcosis.[11] It has been found that CD4+ T-cell plays a critical role on immune responses to C. neoformans.[1213] Th1 cytokines, such as interleukin (IL)-2, IL-12, interferon-γ, and tumor necrosis factor-α, orchestrate neutrophils and dendritic cells and activate macrophages to promote clearance of the pathogen.[14] Some studies figured out that cryptococcal meningoencephalitis is one of the most common disseminated fungal infections in HIV patients and the third most common invasive fungal infection in patients with organ transplants.[81516] Otherwise, mounting evidence showed that the prevalence of cryptococcosis in immunocompetent hosts is increasing.[1516] Then, pulmonary cryptococcosis is the leading type of cryptococcosis in non-HIV patients.[6817] Baddley et al. found that, among 166 HIV-negative patients with pulmonary cryptococcosis, 122 had pulmonary infection only and 44 had pulmonary plus extrapulmonary infection.[17] Some studies showed that patients with pulmonary cryptococcosis were frequently symptomless or with mild symptoms.[689] Then, it is also wildly regarded that the diagnosis of pulmonary cryptococcosis is challenging. At present, serum CrAg screening has become a useful tool in cryptococcosis diagnosis.[181920] High serum CrAg titer was considered to be associated with the load of fungi.[21] Meanwhile, serum CrAg also was used to monitor the recrudescence.[21] Nevertheless, we noticed that having negative serum CrAg is not rare among patients with pulmonary cryptococcosis, which was proven by biopsy later, in our clinical practices. Therefore, the characteristics of patients with and without serum CrAg test positive were worthy to be evaluated. In our study, 114 patients with pulmonary cryptococcosis, proven by PTNB or postoperative biopsy, were screened and 85 patients were enrolled, 56 in CrAg+ group and 29 in CrAg− group. According to our results, we found that male (60.7% in CrAg+ group and 55.2% in CrAg− group) was slightly more than female and cough and expectoration were two most common symptoms [Table 1]. Interestingly, we also noticed that fever was rare in these patients, indicating that fever probably would be used as a symptom for exclusive diagnosis. Moreover, clubbing finger was not observed in our study, which is an important and typical sign of lung cancer, chronic tuberculosis, and other lung diseases. Kohno et al. also showed that only 3% (2 in 67) of non-HIV patients with pulmonary cryptococcosis had fever.[6] Since immunological status and underlying conditions are critical in fungal infection, the underlying status of patients was analyzed in our study. We found that only 17.9% in CrAg+ group and 17.2% in CrAg− group were complicated with underlying diseases. Meanwhile, our data showed that T2DM and kidney transplantation were more common than other diseases [Table 1]. In our study, laboratory findings, including Hb, PLT, WBC, neutrophils, and plasma PCT level, were analyzed. According to our data, no special change was observed between two groups, indicating that no severe systematical inflammatory response was stimulated in immunological systems. Moreover, these can partially explain the reason of symptomless or mild symptoms in pulmonary cryptococcosis. Therefore, further studies should be carried out to elucidate the underlying mechanism of the immune escape in pulmonary cryptococcosis. Then, these results suggested that pulmonary cryptococcosis showed untypical symptoms and laboratory findings, particularly without apparently infection features, in clinical practices. Moreover, most non-HIV adult patients with pulmonary cryptococcosis were not combined with underlying diseases. Therefore, we presumed that underlying diseases play a limited role in the pathogenesis of pulmonary cryptococcosis in non-HIV adult patients.

In light of most of the pulmonary cryptococcosis patients without symptom or with mild respiratory symptoms, they were accidentally found by routine chest X-ray check.[6789] In radiography, some studies reported that chest radiological findings of pulmonary cryptococcosis were untypical and varied.[62223] Many studies found that pulmonary nodules and mass were more common.[6824] Lindell et al. found that the most common findings of computed tomography (CT) scan were multiple, small, well-defined, and smoothly marinated pulmonary nodules in immunocompetent patients with pulmonary cryptococcosis.[24] According to our data, we figured out that single or multiple nodules and mass were most common chest radiological findings. Meanwhile, ground-glass attenuation, infiltration, and consolidation were not rare. However, we also noticed that cavity, pleural effusion, and interstitial changes were seldom. Then, in the current study, we divided the CT findings into two conditions, diffuse extent lesion and limited extent lesion. Lesions in multiple lobes, multiple lesions in a single lobe, and the lesion diameter >3 cm in a single lobe were defined as diffuse extent lesion. Moreover, the lesion (the diameter <3 cm) in a single lobe was defined as limited extent lesion. Our data showed that diffuse extent lesion was more common in CrAg+ group and limited extent lesion was more frequently observed in CrAg− group. Then, we speculated that there should be a positive association between the extent lesion and the pathogen loads in the lung. Therefore, our results indicated that serum CrAg was valuable for extensive extent lesion in non-HIV adult patients with pulmonary cryptococcosis, whereas serum CrAg negative with limited extent lesion would not preclude the diagnosis of pulmonary cryptococcosis.

In addition, according to our data, we showed that pulmonary cryptococcosis was more common than cryptococcal meningoencephalitis and disseminated cryptococcosis in adult patients without HIV infection. However, due to our limited sample size and study centers, more data should be collected in future.

In conclusion, in the current study, our data indicated that, among patients with suspected pulmonary cryptococcosis, extensive pulmonary lesion combined with a negative serum CrAg is highly suggestive of an alternate diagnosis, whereas, among patients with limited pulmonary lesion combined with a negative serum, CrAg does not preclude the diagnosis of pulmonary cryptococcosis.

Financial support and sponsorship

This work was supported by the grants from Guangzhou Programs for Natural Science Foundation of Guangdong Province (No. 201707010282), Scientific Research Project of Guangzhou (No. 2017A030310286), the National Natural Science Foundation of China (No. 81670071), and Science and Technology Planning Project of Guangdong Province (No. 2014A020212627).

Conflicts of interest

There are no conflicts of interest.