Infective patterns of cryptococcosis in patients with connective tissue disease: a retrospective study

To explore the clinical features and associated factors of cryptococcosis in patients with connective tissue disease (CTD) from Southern China. Demographic and clinical data were collected between 2007 and 2018. Associated factors were analyzed by logistic regression analysis. A total of 6809 inpatients with CTD were included. Cryptococcosis was diagnosed in 30 patients (prevalence, 0.4%). Cryptococcosis was predominant in patients with ANCA-associated vasculitis (AAV) (prevalence, 6/530, 1.1%). Lung was commonly involved (18/30, 60.0%), followed by meninges (6/30, 20.0%), blood stream (5/30, 16.7%), and disseminated cryptococcosis (involved blood stream and meninges) (1/30, 3.3%). Infiltrates (10/18, 55.6%) and small nodules (8/18, 44.4%) were the main radiographic manifestation of pulmonary cryptococcosis (PC). The positive rate of serum cryptococcal antigen (CrAg) in patients with PC was 88.2%. Cryptococcus spp. were found in 75% (3/4) patients who underwent lung biopsy. Most of the patients with cryptococcal meningitis (CM) had elevated cerebrospinal fluid (CSF) opening pressure (6/7, 85.7%) and decreased CSF glucose level (5/7, 71.4%). Positive blood culture confirmed the diagnosis of cryptococcal sepsis (CS). Three patients died (10.0%), including one with CM and two with PC. Multivariate logistic regression analysis showed that accumulated dose of glucocorticoid (GC) [odds ratio (OR) = 1.42, 95% confidence interval (CI) 1.04–1.93, P = 0.03] was associated with cryptococcosis in patients with CTD. Cryptococcosis develops in various organs. Typical radiological manifestation accompanied with positive serum CrAg provides helpful clues for the diagnosis. Lumbar puncture is a critical diagnostic method to distinguish CM. The accumulated dose of GC is associated with cryptococcosis in patients with CTD. Key Points • Pulmonary cryptococcosis is suspected if pulmonary nodules adjacent to the pleura are present, with serum CrAg positive. • Cryptococcal meningitis has insidious onset and the diagnosis mainly depends on lumber puncture. • Cryptococcal sepsis is not rare and needs timely blood culture in suspected patients. Key Points • Pulmonary cryptococcosis is suspected if pulmonary nodules adjacent to the pleura are present, with serum CrAg positive. • Cryptococcal meningitis has insidious onset and the diagnosis mainly depends on lumber puncture. • Cryptococcal sepsis is not rare and needs timely blood culture in suspected patients.


Introduction
Infection is one of the major threats to patients with connective tissue disease (CTD) because of the immune disturbance and immunosuppressive therapy [1,2]. As one of the opportunistic infections, invasive fungal disease (IFD) is a matter of concern in immunocompromised hosts. Our previous study found that approximately 1.1% of the lupus patients develops IFD [3]. Aspergillus spp., Candida spp., and Cryptococcus spp. are the common causative agents. However, due to the limitation of sample size, only 15 cases of cryptococcosis in SLE were analyzed. Afterwards, we investigated the specific features of different IFD. In a study enrolling 6911 CTD patients, we found that aspergillosis continues to be a major infectious pattern and the prevalence is the highest in patients with ANCA-associated vasculitis (AAV) [4]. In order to determine clinical characteristics of IFD in patients with CTD, we constructed an in-depth study, focusing on cryptococcosis. We included patients with other CTD besides SLE in order to identify the most likely associated factors with cryptococcosis.
Until recently, researches on cryptococcosis mainly focus on patients with acquired immune deficiency syndrome (AIDS) [5]. Although a number of studies reported that patients with CTD display immune disorders and the prolonged use of glucocorticoids (GC) and immunosuppressants increases the risk of cryptococcosis [6], most of these studies only investigated one of the subcategories in one specific CTD. For example, the incidence rate of cryptococcal meningitis in patients with systemic lupus erythematosus (SLE) is nearly 0.5% [7]. Approximately 0.2% of the patients with rheumatoid arthritis (RA) complicate with pulmonary, central nervous system (CNS) or cutaneous cryptococcosis [8]. Cryptococcosis in other CTD such as systemic vasculitis was only reported in scattered cases [9,10]. The overall situation of cryptococcosis in patients with CTD is not well elucidated. Patients with cryptococcosis could be asymptomatic or develop organ failure based on the infected species and host immunity. Insidious disease onset and delay treatment results in poor prognosis [11]. Therefore, to determine the epidemiological and clinical features of cryptococcosis in patients with CTD might facilitate early recognition and treatment that may reduce mortality.
In this study, we conducted a retrospective analysis, aiming to identify the epidemiology, clinical profile, and risk factors of cryptococcosis in patients with CTD from Southern China.

Study design
We performed a retrospective study on the inpatients (age ≥ 14 years) from the First Affiliated Hospital of Sun Yat-Sen University from January 1, 2007, to December 31, 2018. The ICD-10 coding of discharged diagnoses was used to identify patients with CTD and cryptococcosis (relevant ICD-10 coding in Appendix Table 6). SLE was established using the 1997 American College of Rheumatology classification criteria [12]. The Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score was used to determine disease activity of SLE [13]. ANCAassociated vasculitis (AAV) was determined according to the definitions of the Chapel Hill nomenclature [14,15]. Disease activity of AAV was evaluated by Birmingham Vasculitis Activity Score (BVAS) [16]. RA was diagnosed based on the 1987 ACR revised criteria for the classification of rheumatoid arthritis [17]. RA disease activity was measured by Disease Activity Score of 28 joints-C-reactive protein (DAS28-CRP) [18]. Inflammatory myopathy (IM) was established referring to the classification criteria proposed by Bohan [19]. The diagnosis of systemic sclerosis (SSc) was conducted based on 1980 ARA classification criteria for SSc [20]. Primary Sjogren syndrome (SS) was defined according to the American-European Consensus Group classification criteria for SS [21]. Mixed connective tissue disease (MCTD) was considered based on the Sharp criteria [22]. Pediatric patients were not included considering they have different physical characteristics compared with adults. Patients with incomplete data were excluded. Patients with CTD but no infection during the same period of hospitalization were considered as potential controls and divided by different CTD. Age, gender, subtypes of CTD, and disease duration were analyzed in the infection group. Controls who met the following criteria: (i) age: average age of infection group ± 5 years, (ii) disease duration: median disease duration of infection group ± 1 year were regrouped. Then the control patients were chosen randomly from age/duration-matching subgroups in different CTD. The total number of controls verse cases was set as 4:1. Finally, 80 patients with SLE, 24 patients with AAV, and 16 patients with RA were chosen as controls.

Case definition
Cryptococcosis was determined using the EORTC/MSG 2008 Guidelines [23] and classified into three categories as proven, probable, and possible. In the interest of capturing cases which were most likely to be truly cryptococcosis, we only included proven and probable cases in this research.
Pulmonary cryptococcosis (PC) was diagnosed on the basic of (i) presence of one of the computed tomography (CT) signs (dense, well-circumscribed lesions with or without a halo sign, or cavity); (ii) Cryptococcus spp. in sputum, bronchoalveolar lavage fluid (BALF) or serum cryptococcal antigen (CrAg) positive; (iii) histopathologic examination of lung tissue finding Cryptococcus spp. or recovery of Cryptococcus spp. in pleural fluid culture. Patients with CTD having criteria (iii) were considered as proven cases. Patients with CTD satisfying criteria (i) and (ii) were considered as probable cases. Patients presenting criteria (i) only were classified as possible cases. Cryptococcal meningitis (CM) was diagnosed as proven if Cryptococcus spp. were found in cerebrospinal fluid (CSF). Probable case was considered if the patients with CTD presented meningeal enhancement or other suggestive features in cranial magnetic resonance imaging (MRI) or CT and positive CrAg test in serum or CSF sample. Patients with cranial radiographic manifestation only were considered as possible CM. Cryptococcal sepsis (CS) was established if blood culture yields Cryptococcus spp.

Outcome
All the patients were followed until discharge. Treatment outcome was classified into two categories: improvement and progression. Improvement was defined as clinical symptoms and radiographic imaging partially resolved. Progressive disease was defined as clinical symptoms and radiographic imaging worsened.

Clinical variables
Demographic and clinical data were collected from medical records. Age was grouped according to the World Health Organization (WHO) classification: young people (age ≤ 44 years), middle age (age 45-59 years), and elderly (age ≥ 60 years) [24]. Clinical characteristics of cryptococcosis included symptoms and signs, sites of infection, and treatment regimen. The degree of fever was classified as mild (37.1-38°C), moderate (38.1-39°C), and high (> 39°C). Cryptococcal capsule polyglycan antigen was detected by latex agglutination (LA) test. Specimen culture and biopsy finding were recorded if available. Medication history included daily dose of glucocorticoid (GC) and the use of immunosuppressive agents within the 3 months prior to cryptococcosis.

Statistical analysis
The descriptive variables included mean (standard deviation, SD) or median (interquartile range, IQR) depending on the distribution of continuous variables and number (percentage) for qualitative variables. Between-group comparison was evaluated with chi-square test or Fisher exact test for categorical variables and Student's t test for continuous variables with normal distribution. Between-group comparison was evaluated using the Mann-Whitney U test for continuous variables with non-normal distribution. Variables with clinical significance and P value < 0.10 from the univariate analysis were considered as candidates for the multivariate analysis. The multivariate analysis was preceded by a stepwise forward procedure to identify the factors associated with cryptococcosis. Associated factors were reported with P values, odds ratio (OR), and 95% confidence interval (CI). Correlation between disease activity and infection was evaluated by Spearman's correlation analysis. The level of significance was set at P < 0.05. All statistical analysis was performed with the SPSS 19.0 statistical package (SPSS Inc., Chicago, IL, USA).

Cryptococcal sepsis
Cryptococcal sepsis (CS) occurred in 6 (20.0%) female lupus patients, and one of them also developed CM. The average (SD) age was 32.3 ± 8.9 years. The median (IQR) duration of CTD was 6.0 (6.0, 24.0) months. The median (IQR) time from symptoms onset to diagnosis was 6.0 (5.0, 7.0) days. Most of the patients (5/6, 83.3%) had high fever. Prior to CS, one patient had skin ulcer, and another one was complicated with continuous ambulatory peritoneal dialysis (CAPD)-related peritonitis. Diagnosis was confirmed with positive blood culture obtained when the patients presented high fever. Three strains of C. neoformans were identified in blood culture. The positivity of serum CrAg was 100% (5/5).

Treatment and prognosis
The median (IQR) follow-up was 15.0 (10.0, 24.0) days. Intravenous fluconazole (600 mg/day) was given to 7 patients with PC, and intravenous voriconazole (400 mg twice a day on day 1, and 200 mg twice a day from day 2) to 2 patients. Oral fluconazole was given to 5 patients with reduced dose of 300 mg/day due to impaired kidney function [creatinine clearance rate (Ccr) ranged from 17.3-29.8 ml/min]. Three patients were treated with intravenous fluconazole (600 mg/day) after hemodialysis. One patient with PC received surgery and subsequent oral fluconazole (200 mg/day). Five patients having CM received combined therapy with intravenous amphotericin B and flucytosine (100 mg/kg/day). Since conventional amphotericin B was used, we prescribed the medicine from a low dose (5 mg/day), and escalated by 5 mg/d to 0.5-0.7 mg/kg/ day, following the dose escalation strategy recommended in drug instruction in order to observe the infusion-related acute toxicity and dose-limiting toxicity. One patient with CM was treated with oral fluconazole (400 mg/day) due to decreased Ccr (25.0 ml/min).
Two patients with CS and one patient with CM and CS were treated with amphotericin B and flucytosine. Treatment algorithm was similar to that of CM. Two patients with CS were given amphotericin B alone due to thrombocytopenia prior to antifungal therapy. One patient with CS was treated with oral fluconazole (400 mg/day) due to decreased Ccr (12.6 ml/min).
The mortality rate in CTD patients with cryptococcosis was 10.0% (3/30). The underlying diseases were SLE (n = 1), RA (n = 1), and AAV (n = 1). The mortality rate of PC and CM was 11.1% (2/18) and 16.7% (1/6), respectively. No patient with CS died. The duration from disease onset to diagnosis was 30, 60 and 7 days in three patients, respectively. All the deceased patients (2 females, 1 male) died of co-infection with fungi and bacteria.

Comparison between CTD patients with and without cryptococcosis
A comparison between CTD patients with and without cryptococcosis is shown in Table 4. The serum IgG level was decreased in patients with cryptococcosis (11.0 g/l vs 13.3 g/l, P = 0.001), while CRP level increased (23.3 mg/l vs 2.5 mg/l, P < 0.001). The accumulated dose of GC within the 3 months before cryptococcosis onset was significantly higher in patients in infection group than control group (0.9 g vs 0.2 g, P = 0.01). Multivariate regression analysis showed that high CRP level was associated with cryptococcosis (OR 1.06, 95% CI 1.03-1.09, P < 0.001). Accumulated dose of GC increased the risk of cryptococcosis (OR 1.42, 95% CI 1.04-1.93, P = 0.03) ( Table 5). Further analysis was performed to determine the relation between cryptococcosis and disease activity. The SLEDAI score in patients with SLE was 6.1 ± 2.1 in infection group and 6.5 ± 3.3 in control group (P = 0.65). The correlation between SLEDAI and cryptococcal infection was not significant (correlated coefficient 0.01, P = 0.95). BVAS score in patients with AAV was 8.0 ± 5.8 in infection group and 9.1 ± 6.0 in control group (P = 0.69). The correlation between active AAV and cryptococcal infection was not significant (correlated coefficient − 0.04, P = 0.84). Disease activity in RA, evaluated by DAS28-CRP, was 3.6 ± 1.7 in infection group and 5.3 ± 1.5 in control group (P = 0.07). The correlation between DAS28-CRP and cryptococcal infection was not significant (correlated coefficient − 0.35, P = 0.13).

Discussion
In the current research, we analyzed the infectious profile of cryptococcosis in patients with CTD from Southern China. Cryptococcosis is a common complication in patients with CTD. The diagnosis of PC relies on pulmonary CT scan, serum CrAg detection and lung biopsy if possible. Lumbar puncture and blood culture are reliable methods to diagnose CM and CS. C. neoformans is the predominant species to cause cryptococcosis in our study. Our results are in consistent with other were dyed purple-red by Periodic acid Schiff (PAS) staining (black arrows) within multinucleated macrophages (× 400). c A large number of Cryptococcus spp. (dyed black, green arrows) were seen on periodic acid silver methenamine (PASM) staining (400×) studies conducted in the same or nearby regions, which reported that C. neoformans are responsible for 66.7% and 100% of the cryptococcosis [8,25]. The annual incidence of cryptococcosis ranges from 6.6 cases per million persons in Australia to 15.6 per 100,000 persons in South Africa [26,27]. Although the estimates from mainland China are quite limited, the number of reported cryptococcosis is increasing generally [28]. A population-based epidemiological study conducted in Taiwan reported the annual incidence of cryptococcal meningitis from 2000 to 2007 was 4.7 cases per million persons [29]. The incidence rate of cryptococcosis was 5~10% in immunocompromised hosts and 30% in AIDS patients [30]. The prevalence of cryptococcosis in CTD in our study (0.5~1.1%) is comparable with previous reports in patients with SLE or RA (0.2~1.0%) [7,8,25], lower than AIDS population but significantly higher than the general population. Therefore, to analyze the clinical characteristics of cryptococcal infection in patients with CTD is necessary.
Cryptococcosis could develop in various organs. The subcategory of cryptococcosis seems to differ in patients with different comorbidities. Patients with HIV or organ transplantation are prone to develop cryptococcal meningoencephalitis [31], while patients with CTD, as reported in the current study, are more likely to have PC. In line with our findings, a previous study on RA [8], also found that lung is the most frequently involved, and the prevalence of PC is approximately 60.0% in both previous and current studies. Hemoptysis, which is common in pulmonary aspergillosis and lung cancer, is rare in cryptococcosis. Nodules, especially small nodules adjacent to the pleura are common radiographic manifestation [32], while cavitary lesions are   CrAg cryptococcal antigen, CSF cerebrospinal fluid, IQR interquartile range, SD standard deviation less frequent compared with that in pulmonary aspergillosis [33]. Serum CrAg is a useful predictor for cryptococcal infection [34], and pathological staining is a productive method to distinguish Cryptococcus spp. Generally, the diagnosis of PC depends on the assessment of clinical symptoms, radiographic manifestation, and etiological identification. PC should be taken into consideration in patients having pulmonary infiltrates or small nodules but inadequate response to antibiotics. Serum CrAg provides valuable clues for the diagnosis of cryptococcosis. Lung biopsy is suggested if diagnosis is still not established through the aforementioned procedures. CM is a major pattern of cryptococcal infection in our study. Among three subcategories of cryptococcosis, both the duration of CTD and the diagnostic time are the longest in patients with CM. Insidious onset of the disease with nonspecific symptoms usually delays the diagnosis. As reported, 38.2% of CM in lupus patients are misdiagnosed as nonfungal infection or active disease and the case-fatality rate is as high as 23.6% [7]. Therefore, applying a rapid and sensitive method for early diagnosis is important. The common radiographic manifestations of CM are leptomeningeal enhancement (42.1~81.1%) and ventricular dilation (14.7~42.9%) [35,36]. Cystic lesions tend to localize in the basal ganglia or periventricular areas and appear in 8.3~19.4% of patients in a review literature including 84 HIV and 81 non-HIV patients [35]. However, the estimates are quite different in another single-center Chinese study. Among 157 non-HIV patients with CM, pseudocysts/cryptococcomas were found in 1.9% and 2.1% in immunocompetent and immunocompromised patients, respectively [36]. The incidence of cryptococcomas/ cerebral abscess in patients with CTD is reported to be 12.5~16.7% [25,37]. It is worth noting, however, that only a very small number of patients (less than 10) with CTD were included. Besides, cryptococcomas, pseudocysts, and cerebral abscess are not always well discriminated in the literatures and could be used interchangeably, which results in disparity. Since the imaging findings are usually non-specific, and approximately 2~26.3% of the non-HIV patients with CM have normal neuroimaging [35,36], lumber puncture appears to be a reliable and necessary method to diagnose CM. Remarkably elevated CSF opening pressure with decreased glucose level implies CM. Diagnosis is confirmed by the isolation of Cryptococcus spp. in CSF by Indian ink or culture. Previous study suggested that CrAg in CSF is an effective tool to distinguish CM, since CSF culture usually takes at least 72 h for Cryptococcus spp. to grow [38]. CrAg detection in CSF provides higher sensitivity and specificity compared with India ink stains and CSF culture [39]. High titers of CrAg in CSF also predict poor neurological outcome [37]. However, due to technological constraints, CrAg was not detected in CSF in our study. We believe that the application of CrAg detection in CSF would improve the timeliness and accuracy of diagnosis in patients with CTD in our center.
CS is not rare in patients with CTD and accounted for 20.0% of the cases in the current research. Previous reports indicated that the prevalence of CS in the Chinese SLE patients is 11.1-19.6% [6,40]. The characteristics of CS are less discussed. Of note, two patients had skin infection or conduit communicating with the outer space. Such patients are risky for CS once exposed to the contaminated air. The isolation of Cryptococcus spp. in blood is the critical and unique method to confirm CS. Therefore, blood culture in the case of high fever is necessary especially in patients with skin lesions.
Generally, the prognosis of cryptococcosis is relatively favorable. Most of the patients improved after effective treatment [41,42]. In order to prevent severe adverse effect of conventional amphotericin B, we applied a dose-escalation strategy and it usually takes a week to reach the therapeutic dose. The delay of time to stable plasma concentration could have negative impact on therapeutic efficacy. An alternative to lipid formulations should be considered if the patient could afford the high expense.
Our research had certain limitations. The prevalence of cryptococcosis in patients with CTD could be underestimated since only inpatients from a tertiary medical center were included. Besides, clinical experience originating from one center limits its generalizability to other population. The retrospective nature has limited value in studying treatment response and long-term prognosis.

Conclusions
Cryptococcosis is a common opportunistic infection in patients with CTD. It can develop in various organs including lung, brain, and blood stream. Typical manifestation accompanied with positive serum CrAg suggests PC. CM usually presents insidious disease onset. Lumbar puncture is a critical diagnostic method. CS is not rare and blood culture is reliable and necessary in suspected patients. The accumulated dose of GC is associated with cryptococcosis in patients with CTD. AAV ANCA-associated vasculitis, CTD connective tissue disease, ICD-10 International Classification of Diseases-10, IM inflammatory myopathy, MCTD mixed connective tissue disease, pSS primary Sjogren syndrome, RA rheumatoid arthritis, SLE systemic lupus erythematosus, SSc systemic sclerosis