Diagnostic value of serum Aspergillus IgG antibody for pulmonary aspergillosis in non-agranulocytic patients

Background: At present, serum Aspergillus IgG and IgM antibodies are mainly used in the diagnosis of chronic pulmonary aspergillosis (CPA), but the diagnostic value in invasive pulmonary aspergillosis (IPA) in non-agranulocytic patients is still unclear. The aim of this study was to investigate the diagnostic value of serum Aspergillus IgG and IgM antibody detection for pulmonary aspergillosis in non-agranulocytic patients.. Methods: 58 cases of pulmonary aspergillosis (37 IPA and 21 CPA cases), 15 cases of bacterial pneumonia and 50 cases of healthy control group were collected. Serum G test and GM test were performed, and Aspergillus IgG and IgM antibody were detected in all patients. The sensitivity and specificity, cut off value and AUC of Aspergillus IgG and IgM antibodies were further obtained by the ROC curves. Results: The positive rates of G test and Aspergillus IgG antibody in pulmonary aspergillosis group were significantly higher compared with bacterial pneumonia group or healthy group ( P = 0.015 and P < 0.0001, respectively). Aspergillus IgG antibody can preferably distinguish CPA from bacterial pneumonia and healthy controls (sensitivity = 0.952, specificity = 0.692, cut off value = 75.46, AUC = 0.873). However, the performance of it was poor in distinguishing IPA from CPA, healthy group and pneumonia group (AUC < 0.7). Conclusions: Serum Aspergillus IgG has certain clinical value in the diagnosis of pulmonary aspergillosis in non-agranulocytic patients.

years, the incidence of pulmonary aspergillosis in non-granulocytic patients has increased with the aging, the increase of chronic diseases, the use of broad-spectrum antibiotics, hormones, immunosuppressive drugs and invasive operations [1,2]. Moreover, the clinical manifestations of these patients lack specificity, often the diagnosis is difficult. As a result, the patient's treatment is delayed, which affects the prognosis of patients.
Pulmonary aspergillosis can be divided into different types in the light of different classification criteria. According to the onset time, course and symptoms, it can be divided into acute pulmonary aspergillosis (APA), subacute pulmonary aspergillosis and chronic pulmonary aspergillosis (CPA). Furthermore, CPA is usually seen in immunocompetent individuals with underlying respiratory disorders, and the prevalence of CPA worldwide is about 3 million [3]. Unfortunately, respiratory physicians may not detect CPA until the disease progresses to an advanced stage, owing to the lack of specific clinical manifestations. More seriously, without timely diagnosis and long-term antifungal treatment, the 5-year mortality rate is nearly 80% [4]. Based on the forms and causes of aspergillosis, pulmonary aspergillosis can be divided into three types: invasive, allergic and parasitic, which reflect different immune statuses of the host. Among them, invasive pulmonary aspergillosis (IPA) has become a common type of severe pneumonia with the highest mortality, and one of the important reasons is that it is difficult to be diagnosed [5]. In addition, patients with agranulocytosis are considered to be the predominant group of IPA, and relevant international guidelines for diagnosis and treatment also focus on this group [6].
It is well known that serum Aspergillus IgG and IgM antibodies are mainly used in the clinical diagnosis of CPA [7]. Synchronously, serum G test and GM test, as non-invasive diagnostic methods, are mainly used for the diagnosis of IPA in agranulocytic patients, but they are not sensitive to non-agranulocytic patients. In this study, we explored the diagnostic value of G test, GM test, serum Aspergillus IgG and IgM antibodies detection for IPA and CPA in non-agranulocytic patients.

Methods
Patients and Data Collection 58 pulmonary aspergillosis cases in non-agranulocytic patients were enrolled which were admitted to Tianjin Chest Hospital from July 2017 to July 2018, and possible IPA cases and cases of allergic bronchopulmonary aspergillosis were excluded. The diagnostic criteria referred to the consensus of experts in diagnosis and treatment of pulmonary mycosis and the criteria of the European Organization for Research and Treatment of Cancer (EORTC) [8,9]. During the same period, 15 cases of bacterial pneumonia and 50 cases of health examination were treated as control groups. The sex and age of control groups had no significant difference compared with pulmonary aspergillosis group. Moreover, the following data were collected: demographic data (age, gender, weight), serum indexes, imaging features, biochemical indicators, bacterial and fungal culture results, bronchoscopic findings, and the treatment outcomes. In addition, all participants has signed the informed consent voluntarily, and the study has been approved by the ethics committee of Tianjin Chest Hospital (2018KY-009-01). Serological Testing 5 ml venous blood were drawn before using any antibiotics. Serum was separated from the blood for tests directly or stored frozen at −80°C. The serum (1,3)-β-D-glucan test (G test) was conducted with a chromogenic method [10]. In brief, 5 μl serum samples were firstly Statistical Analysis SPSS 21.0 software was used for statistical analysis. Comparison between two groups were performed by chi-square test. Fisher's test results were used when the sample size was small and the theoretical number was small. Mann-Whitney U test was used in the course of disease, age and serum indicators except lymphocyte count indicators. Independent sample t test was used for lymphocyte count indicators. The sensitivity, specificity and optimal threshold were determined by using the receiver operating characteristic curve (ROC curve). The standard of this study was P < 0.05 with statistical difference.

Patient Characteristics
Characteristics of the 58 pulmonary aspergillosis patients were shown in Table 1

Characteristics Comparison between IPA and CPA Cases
Clinical features between IPA and CPA cases were compared and exhibited in Table 2, including microbiological findings, clinical symptoms, thoracic CT signs, involving lobes of lung, and serum indexes. It was obvious from Table 2 that the course of disease was longer in CPA cases than IPA cases. Some clinical symptoms, such as fever, dyspnoea and haemoptysis, were very different between IPA and CPA cases (P < 0.05). Ulteriorly, there were observable differences between above two groups in thoracic CT signs of infiltrates, air crescent sign and ground-glass attenuation, lung lobes of right middle, right lower and left upper, serum indexes of LDH, albumin, PCT levels and lymphocyte count (P < 0.05, Table 2).

Results of Serum G test, GM test, Aspergillus IgG and IgM Antibodies in Each Group
Results of serum G test, Aspergillus IgG antibody, Aspergillus IgM antibody and GM test were listed in Table 3A-C among different groups. Primitively, positive rates of above serum indexes were counted among pulmonary aspergillosis, bacterial pneumonia and healthy groups, and Table 3A was the statistical result. It was illustrated that the positive rates of serum G test and Aspergillus IgG antibody were notable higher in pulmonary aspergillosis group than bacterial pneumonia and healthy groups (P = 0.015 and < 0.0001, respectively). Afterwards, in order to study whether different types of pulmonary aspergillosis could be distinguished, the pulmonary aspergillosis group was divided into IPA and CPA groups according to the disease type. Table 3B was the comparison result among IPA, CPA, bacterial pneumonia and healthy groups, and Table 3C shown comparison between IPA and CPA groups. Besides G test and Aspergillus IgG antibody, the positive rate of GM test also showed notable differences among IPA, CPA, bacterial pneumonia and healthy groups (P = 0.022) (Table 3B). Nevertheless, G test and Aspergillus IgG antibody were no markedly statistical difference between IPA and CPA groups (P ≥ 0.5), and the positive rate of GM test statistically evident difference (P = 0.04) (Table 3C).

ROC Curves of Serum Aspergillus IgG Antibody in Different Groups
The ROC curves of Aspergillus IgG antibody in different groups were drawn. Fig. 1 A-F displayed ROC curves of Aspergillus IgG antibody with remarkable significance (P < 0.05), and the cutoff value (sensitivity, specificity) and the area under curve (AUC) were also shown. It was revealed that Aspergillus IgG antibody with the highest specificity (0.952) when IPA group compared with CPA groups (Fig. 1B), with the highest sensitivity (0.952) when CPA group compared with IPA, bacterial pneumonia and healthy groups (Fig. 1F), and with both the highest AUC (0.873) and the highest sensitivity (0.952) when CPA group compared with bacterial pneumonia and healthy groups (Fig. 1D). Furthermore, the AUC value was bigger in Fig. 1D than that of Fig. 1C, so as to in Fig. 1F than that of Fig. 1E.
That was, serum Aspergillus IgG antibody had a better performance for distinguish CPA than IPA.

Discussion
Although pulmonary aspergillosis in non-agranulocytic patients has increased with the development of society, the frequency remains low relative to that in agranulocytic patients. So far, few data are available in non-agranulocytic cases, and most of them are case reports [11][12][13][14]. Consequently, more cases and more studies are urgently needed to understand non-agranulocytic pulmonary aspergillosis, so as to provide more references or clues for the diagnosis and treatment of the disease. In this article, 58 cases were reported, and the sample size was rare and higher. IPA is a life-threatening infection in patients mainly with prolonged neutropenia. One clinical challenge of non-agranulocytic IPA cases is the frequently lack of specific clinical features, especially in those without underlying disease [15]. In our study, we roundly compared clinical features between IPA and CPA cases with relevant diagnostic methods commonly used in clinic (Table 2), including microbial cultivation, thoracic CT and serum detection. Some special characteristics for IPA were spotted, such as shorter disease course, frequent infiltrates, special lobe of lung, lower serum albumin level, which might be used for differential diagnosis or auxiliary diagnosis.
The diagnosis gold standard of pulmonary aspergillosis mainly relies on chest imaging, microbial culture and histopathological examination. However, the imaging manifestations are poor in specificity for non-agranulocytic patients, and the phenomenons of "the same disease with different image, and the different disease with same image" exist [16,17].
As for microbiological and histopathological examination, it is difficult to obtain pathological specimens, positive rate of culture is low, and possibly contaminated and colonized. Therefore, the clinical diagnosis of non-agranulocytic pulmonary aspergillosis is difficult, and it is not always feasible to obtain histo-or cytopathological demonstration of the fungus in order to meet the gold standard [18]. As a non-invasive diagnostic method of pulmonary mycosis, the detection of serum antigens and antibodies has attracted more and more attention. G test and GM test are mainly used for the clinical diagnosis of IPA in agranulocytic patients, but the positive rate of IPA in non-agranulocytic is too low to meet clinical needs [19,20]. For patients with agranulocytosis or severe immunosuppression, it is difficult for the body to produce an immune response. Accordingly, the detection of specific antibodies against Aspergillus is of little significance. With the increase of nonagranulocytosis and non-immunocompromised host, the diagnostic significance of antibody detection for pulmonary aspergillosis needs to be reevaluated. Serum Aspergillus antibody detection is mainly used in the diagnosis of CPA [21,22]. Meanwhile, the diagnostic value of Aspergillus antibody is not clear for IPA in non-agranulocytic patients because of varying results [18]. Additionally, diagnosing chronic pulmonary aspergillosis (CPA) is complicated, and there are limited data available [23]. Here, we compared the performances of G test, GM test, Aspergillus IgG antibody by using serum samples from non-agranulocytic patients with underlying pulmonary aspergillosis diseases, and further subdivided IPA and CPA (Table 3A-C). There are few studies on serum Aspergillus IgM antibody, and its significance in the diagnosis of pulmonary aspergillosis is not clear. This study showed that there was no significant difference in serum Aspergillus IgM antibodies between pulmonary aspergillosis, bacterial pneumonia and healthy people. The reasons may include: 1. IgM is the earliest immunoglobulin produced after infection or immunization. It has strong bactericidal and regulatory effects, but its content in blood is low, half-life is short, and it is susceptible to interference factors. 2. Non-granulocytedeficient hosts may undergo a period of Aspergillus colonization and slow invasion before infection due to their relatively sound immune function. IgM often occurs in the early stage of infection. Therefore, Aspergillus IgG antibody detection is more significant than Aspergillus IgM antibody detection. Our results revealed that Aspergillus IgG antibody reflected the greatest differences among pulmonary aspergillosis (even IPA and CPA subdivision), bacterial pneumonia and healthy group (P < 0.0001) (Table 3 A, B). It was indicated that Aspergillus IgG antibody might a potential diagnostic index for pulmonary aspergillosis in non-agranulocytic patients, and it was further evaluated the performance through ROC curves.
As exhibited in Fig. 1, Aspergillus IgG had notable different in pulmonary aspergillosis (even IPA and CPA subdivision), bacterial pneumonia and healthy group (P < 0.05), and both the specificity and sensitivity were 40.5-95.2% and 58.8-95.2%, and the highest AUC 0.873. Previous studies have shown that the sensitivity and specificity of Aspergillus IgG antibody detection for CPA diagnosis are 75-96% and 97-99% [24]. The specificity and sensitivity were lower than the previous report, it might because that the underlying condition of the research population and the experimental methods are different. Our study further certified that serum Aspergillus IgG antibody had a better performance for distinguish CPA than IPA. From acute invasive infection to chronic consumptive diseases, different types of pulmonary aspergillosis can overlap with each other. Generally, IPA occurs in patients with impaired immune function in varying degrees, while CPA occurs in patients without or with impaired immune function in a lower degree. Therefore, serum Aspergillus antibody levels differ in different types of pulmonary aspergillosis, which is of greater significance to patients with CPA. Above all, we suspected that serum Aspergillus IgG has certain clinical value in the diagnosis of pulmonary aspergillosis in nonagranulocytic patients, especially for non-agranulocytic CPA. Howbeit, it was believed that serum Aspergillus IgG could not replace the traditional isolation and culture of fungi, and should be combined with other diagnostic methods and clinical practice. In addition, further studies were needed to determine the role of Aspergillus specific antibodies in the pathogenesis, diagnosis and treatment of aspergillosis.

Conclusions
In conclusion, serum Aspergillus IgG has certain clinical value in the diagnosis of pulmonary aspergillosis in non-agranulocytic patients.

Declarations
Ethics approval and consent to participate All participants has signed the informed consent voluntarily, and the study has been approved by the ethics committee of Tianjin Chest Hospital (2018KY-009-01).

Consent for publication
Not applicable.

Availability of data and material
All data used during the study are available on reasonable request.