Imaging Features Of Invasive Pulmonary Fungal Infections: A Clinical And Animal Experimental Research

【Background】Fungus broadly exists in the natural word, and has opportunistic pathogenicity to humans. The occurrence and development of fungous disease rely on three condition:The characteristics and quantity of infecting fungus, the type of underlying immune defect, the duration and severity of immune compromise, and the specific epidemiologic or environmental exposure. There are three results from fungus infecting host: 1.The pathogenic fungi is inhibited by inflammatory reaction and phagocytosis on condition of complete immune function.2. The inhaled spores have a propensity to germinate and proliferate in the proximal airways and induced asthma-associated mucosal injury.3.Invasive fungal infections occur on the condition of host immunity compromised. During the past few decades, the population of immunocompromised hosts has expanded enormously, reflecting the increased use of immunosuppressive agents for the treatment of tumors and collagen vascular disease and for the prevention of rejection in organ transplant recipients. In addition, acquired immunodeficiency syndrome (AIDS) has resulted in the existence of many immunocompromised patients. The incidence rate of invasive pulmonary fungal infections (IPFI) has increases year by year, which has been research priorities recently. IPFI typically affects immunosuppressed critically ill patients, and has bad prognosis and high fatality rate. The key to the treatment of pulmonary fungal infections and decrease of mortality rate, is early detection and diagnosis of pulmonary fungal infection, especially IPFI, with timely and effective lines of antifungal therapy. With the image of scientific development, especially in thin spiral CT imaging technology innovation, clearly shows the fine structure of the lungs and microscopic pathology is possible. Imaging method used for early detection and diagnosis of pulmonary fungal infection is present in the field of the important issues. By the impact of immune status of patients, the type and quantity of invasive pathogens, as well as the different routes of infection, imaging findings of IPFI are very complex and little characteristic signs have been found with diagnostic practical value. Therefore, the present study gives two directions to investigate the characteristic imaging findings, mechanism, as well as the develop pattern of IPFI ,including retrospective analysis from clinical cases and establishment of the animal model, in order to improve knowledge of imaging findings in IPFI and elevate early diagnostic accuracy. PartⅠClinical retrospective study of imaging features in invasive pulmonary fungal infection【Objective】To explore the imaging characteristics of invasive pulmonary fungal infection caused by different pathogens and to provide the foundation for the early diagnosis of disease.【Material and Methods】1 Patient data: Clinical and radiological data was from patients with clinical diagnosis or definite diagnosis of invasive pulmonary fungal infection in Changzheng Hospital and Shanghai Public Health Center Clinical Center affiliated to Fudan University from October 2006 to October 2010.2 Inclusion criteria of patients: The diagnosis of IPFI must meet the―Diagnostic criteria and treatment principles of invasive pulmonary fungal infection‖(draft) issued by the Editorial Board of Journal of Internal Medicine in 2006.3 Exclusion criteria of patients:(1)Pneumocystis carinii pneumonia(PCP)is exclusive comply to―Revised Definitions of Invasive Fungal Disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group‖.(2)Fungal infections caused by fungi parasitic or allergies reaction. (3) Concomitant infection in patients with other pathogens (bacteria, mycoplasma, chlamydia, virus,tuberculosis, etc.)4 The parameters of computer tomography(CT) examination: CT scan information of patients is comply to the final diagnosis of pulmonary infection, the CT machine type includes Marconi Mx8000,16-slice MDCT scanner(TOSHIBA Acquilion orSomatom Sensation ) or 64-slice MDCT scanner(GE Light Speed VCT),scanning from the apex of lung to the base of lung with routine exposure dosage. CT enhancement:a total dosage of 70～90ml non-ion iodine agent was administered at a rate of 5ml/sec into opisthenar vein,scanning time are 25～45 and 2～4min after injecting.5 CT image analysis: 2 Chest physician radiologists read films respectively. When opinions are different, coincidence was reached after negotiation .The evaluating contents including the scope, distribution and appearance of the main pathological changes. After the assessment of lung parenchymal abnormalities in pulmonary cryptococcosis, the lung lesions were further classified into four patterns:single nodular or mass, multiple clustered nodular, multiple scattered nodular, and bronchopneumonic patterns. 6 Statistical method: Statistical analysis is get from SPSS16.0 software. Absolute is represented by mean±standard deviation, the relative number by rates, the compare between two groups usingχ2 or Fisher exact test analysis.【Results】1 Clinical Data and Statistics(1)117 cases of patients with IPFI were collected: 34 cases came from Chang Zheng hospital and 83 cases from Shanghai Public Health Center. 101 patients (86.3%) had immunosuppressed factors, including AIDS in36 cases(35.6%), organ or bone marrow transplantation in 28 cases (27.7%) and liver cirrhosis in 24 patients (23.8%),which were common underlying diseases. Mortality of patients in two hospitals was 26.5% and 38.6% respectively, and rates of extrapulmonary dissemination were 20.6% and 33.7% respectively.(2) A total of confirmed or clinical diagnosis was made in pulmonary cryptococcosis in 43 cases, invasive pulmonary aspergillosis in 35 cases , pulmonary candidiasis in 22 cases, Aspergillus and Candida mixed infection in 1 case, pathogens are not clear in 16 cases . Among them 57 patients had confirmed diagnosed, in which lung tissue samples obtained by percutaneous biopsy in 24 cases, by biopsy or surgical thoracotomy in 13 cases, under the guidance of bronchoscopy biopsy in 8 cases, and with positive pleural fluid culture and blood culture in 12 cases. 60 patients was under clinical diagnosis , among which qualified sputum examination positive in 37 cases, bronchial lavage fluid examination or culture positive in 12 cases, blood or pleural fluid samples culture or antigen tested positive in 11 cases.2 Thoracic CT appearances(1)Common CT signs: The most common CT appearances of IPFI were solitary or multiple nodules or mass which found in 88 cases (75.2%), followed by airspace consolidation in 59 cases (50.4%) and ground glass opacity(GGO) in 34 patients (29.1%). Nodules tend to distributed in peripheral zone (P = 0.003), while airspace consolidation tends to locate in lower lung (P = 0.013).It had no significant difference in unilateral or bilateral distribution comparing three type lesions (P = 0.085). The incidence of cavity in nodules (89.3%) was significantly higher in AIDS patients than that of non-AIDS immunosuppressed persons (47.0%, P = 0.000), the incidence of halo sign around nodules was significantly higher in non-AIDS immunosuppressed persons (67.3%) than that of AIDS patients (32.1%) (P=0.010). The rate of multiple lesions in airspace consolidation was significantly higher in immunosuppressed patients(75%) than that in immunocompetent patients (28.6%, P = 0.023). Pleural effusion only occur in immunosuppressed persons, the incidence in non-AIDS immunosuppressed persons (32.1%) was significantly higher than that in AIDS patients (11.1%, P = 0.018).(2) CT appearance in three common IPFI: The most common CT findings in pulmonary cryptococcosis was single or multiple nodules or mass (86.0%), followed by airspace consolidation (16.3%) and GGO(14.0%),including 36 cases with simple lesions (92.3%) and 7 with mixed lesions (7.7%). The most common CT findings in IPA / CNPA was airspace consolidation in 23 cases (65.7%), followed by nodule or mass in 22 cases (62.8%) and ground glass in 12 cases (34.3%), including 15 cases with simple lesions (42.9%) and 20 with mixed lesions (57.1%). The most common CT findings in pulmonary candidiasis was single or multiple nodules or mass (68.1%), followed by airspace consolidation (63.6%) and GGO(45.5%),including 8 cases with simple lesions (36.4%) and 14 with mixed lesions (63.6%).(3) The comparison of CT appearances in three common IPFI: The rate of nodules in multiple clustered pattern in Pulmonary cryptococcosis was significantly higher than that in IPA / CNPA (P = 0.004). The rate of nodules in multiple scattered pattern in IPA / CNPA and in pulmonary candidiasis was significantly higher than that in pulmonary cryptococcosis respectively (P = 0.000, P=0.000).The incidence of air bronchogram in nodules of pulmonary cryptococcosis was significantly higher than that of pulmonary candidiasis. The incidence of cavity in nodules of IPA / CNPA was significantly higher than that of pulmonary candidiasis. The rate of airspace consolidation in IPA / CNPA and in pulmonary candidiasis was significantly higher than that in pulmonary cryptococcosis respectively (P = 0.000, P=0.000). The pleural-based pattern in airspace consolidation of IPA / CNPA was significantly higher than that of pulmonary cryptococcosis (P = 0.001). The incidence of peribroncovascular distribution (P = 0.007) and air bronchogram (P = 0.001)in airspace consolidation of pulmonary cryptococcosis was significantly higher than that of IPA / CNPA.(4)The comparison of CT appearance of pulmonary cryptococcosis in different immune status:The incidence of cavity in nodules (78.9%) in AIDS patients was significantly higher than that in non-AIDS immunosuppressed patients (36.3%, P = 0.047).The incidence of air bronchogram in nodules in immunocompetent patients(71.4%)was significantly higher than that in immunosuppressed persons (23.3%, P = 0.025), which in non-AIDS immunosuppressed patients (71.4%) was also significantly higher than that in AIDS patients (5.3%, P = 0.004).【Conclusion】1. IPFI mainly involves immunosuppressed patients. The most common CT appearance was multiple nodules and airspace consolidation Nodules tend to distributed in peripheral zone, while airspace consolidation tends to locate in lower lung.2. CT findings of IPFI were different in patients with different immune status: AIDS patients tend to form cavity in nodules. Non-AIDS immunosuppressed persons prone to have halo sign around the nodules. Consolidation is prone to form multiple lesions in immunosuppressed patients, and to show single lesion in immunocompetent patients.3.Pulmonary cryptococcosis was common in simple pattern. Single or multiple nodules were the most important CT signs, among which clustered distribution and air bronchogram is the main features different from nodules in other IPFI4. The pattern of nodules in pulmonary cryptococcosis was different , associated with different immune status: cavity is more common in nodules in AIDS patients, while air bronchogram is more common in nodules in immunocompetent patients.5. Mixed pattern is more common in IPA/CNPA and pulmonary candidiasis. The most common CT findings were multiple nodules combined with airspace consolidation. Multiple nodules were tend to in scattered distribution.6. Cavitation particularly air crescent sign was the characteristic sign in nodules of IPA/CNPA. Pleural-based consolidation was the other important sign to differentiate IPA/CNPA from pulmonary cryptococcosis. PartⅡAn animal experi ment of pulmonary candidiasis: The imaging features and developing tendency at the early stage【Objective】To explore the relationship between CT appearance and pathological changes of pulmonary candidiasis at acute stage, by establishing animals model in different immune status and different infective routes.【Methods】1. Experimental animals and materials: Forty New Zealand rabbits and standard strain of Candida albicans ATCC10235.2. Methods to establish animal model: Forty New Zealand rabbits were code for 1-40 number and divided into four groups randomly (A,B,C,D). During the first five days of the experiment, Ara-C at a dose of 440mg/m2 (body surface area) was injected daily via the ear vein in both group A and group B to produce profound and persistent neutropenia. After 6 days, Ara-C was injected on alternative days to maintain a low immune status. The results of blood routine test on day 1 and 6 were compared. On and after day 4, antibiotics were given intravenously via the ear vein in all four groups, including vancomycin, 15mg/kg; ceftazidime, 150mg/kg, daily; gentamycin, 5mg/kg on alternative days to prevent the occurrence of invasive bacterial infections during neutropenia. On the 6th day, 0.2ml C. albicans suspension at 5×108cfu/ml was injected into the trachea of the rabbits in group A and group C through percutaneous tracheal puncture. An equivalent amount of C. albicans suspension was injected intratracheally in group B and group D. Blood culture examination by the ear vein blood samples was done on day1, 3, 7 after inoculation.3. Determination of animal model: Chest CT scan was performed with a 16-slice spiral scanner (Somatom Sensation 16; Siemens Medical Systems, Forchheim, Germany) in both groups before and on day 2, 4, 6, 8, 10, 12 and 14 after inoculation. On day 14 after inoculation, all rabbits were sacrificed if they did not die naturally. Lung tissue samples of all rabbits were obtained by autopsy according to the location of CT findings. All samples were pathologically diagnosed by HE (Hematoxylin-eosin) and PAS (periodic acid-Schiff) staining and microbiologically diagnosed by fungal culture. Liver, kidney and spleen samples were got for fungal culture by the same way.The following diagnostic criteria for Candida pneumonia were adopted in our study: (1) The inoculated C. albicans was identified in the lung tissue samples by fungal culture from Sabouraud's medium; (2) The focus of infection was detected by CT scan; (3) HE staining of the lung tissue samples showed fungal invasive appearance and inflammatory pseudohyphae were found by HE or PAS staining. If the first criterion was confirmed, the inoculation was considered successful. If all above criteria were confirmed, the rabbit model with C. albicans pneumonia was considered to have been established successfully. If the first criterion was confirmed but the second or the third was not meanwhile, it was thought of as the situation of dormant infection with C. albicans field planting in rabbit lungs.4. Statistical method: Statistical analysis is get from SPSS16.0 software. Absolute is represented by mean±standard deviation, the relative number by rates, compare between two groups usingχ2 or Fisher exact test analysis.【Results】1. Modeling results of pulmonary C. albicans infection in immunosuppressed rabbits: 23 rabbit model with pulmonary candidiasis were successfully established, including 8 cases in group A, 7 cases in group B, 2 cases in group C and 6 cases in group D. The modeling success rate in immunosuppressed group (A + B) and the immunocompetent group (C + D) was 75% and 40% respectively (p = 0.025), which in tracheal inoculation group (A + C) and venous inoculation group (B + D) was 50% and 65% respectively (p = 0.337).All rabbits died naturally within 14 days after inoculation. The mortality in the first week after inoculation of tracheal inoculation group (A + C) and venous inoculation group (B + D) was 15% and 70% respectively (P = 0.001). lung tissue culture positive rate was 100% in tracheal inoculation group (A + C) , and kidney fungal culture positive rate was 100% in venous inoculation group (B + D).2. Thin-section CT findings: Abnormalities were detected on thin-section CT scans from day 2 to day 14 after inoculation. The main CT findings were GGO (19/23), which showed diffuse distribution in 7 cases , multiple patchy in 9 cases, focal distribution in 3 cases and had a gradually increasing range;consolidation (10/23), which showed diffuse distribution in 5 cases, multiple patchy in 5 cases, focal distribution in 1 cases ; and multiple nodules (3/13) with diameter less than 1cm,among which 6 cases mainly distributed in the peripheral zone, and gradually progress to the central zone and showed diffuse distribution. 2 cases were first found distributed around the bronchovascular bundles and progress in the bronchial end in one and integration into consolidation in the other. 2 cases of nodules were scattered in the peripheral lung with no obvious trend in progress. Lesions in each group are mixed pattern and mainly in diffuse distribution. Consolidation and GGO were the main CT findings in 2-4 days after inoculation, while histological findings, including hemorrhage, hyperemia, exudation, infla mmatory cell infiltration, interstitial hyperplasia, vascular thrombosis and necrosis; and/or yeast cells or of the lung tissue samples showed fungal invasive appearance and infla mmatory multiple nodules and GGO were 1 week after inoculation. The incidence of nodules was in significant difference between tracheal inoculation group (A + C) and venous inoculation group (B + D) (80%, 15.4%; P = 0.003).3. Pathological findings: 19 areas of GGO seen on CT scans corresponded to the exudative or proliferative phase of diffuse alveolar inflammation or interstitial hyperplasia. Well-defined hemorrhagic infarcts were observed in 9 consolidations, and thrombi were observed in the pulmonary arteriole in 8 of them Histologically, 3(3/10) nodules showed well-defined granuloma, and 4 of which were observed thrombi in the focal necrosis. The spore and/or pseudohypha of C. albicans were found by HE staining in 14 (14/23) cases and PAS staining in 19 (19/23) cases.4. Type of models: Three forms of pulmonary candidiasis were noted. Bronchopulmonary form (4/23) was found in 3 cases in group A and 2 in group C, Embolic form (13/23) was found in 5 cases in group A, 5 in group B and 2 in group C, Capillary-invasive form (6/23) was found in 1 cases in group A, 2 in group B and 3 in group C.【Conclusion】1. The animal model of primary and secondary pulmonary candidiasis can be successfully established by the way of tracheal and intravenous inoculation. The incidence of pulmonary candidiasis associated with the host immune status, and had nothing to do with the infectious route. Two routes of infection were easily lead to multi-system disease, but the intravenous route had more rapid progression of infection.2. Pulmonary candidiasis in rabbit models showed a mixed type lesion with a diffuse distribution. The very early ct appearance was consolidations associate with GGO, which appeared in 2-4 days after inoculation. Mainly appearance at the acute phase was nodules associate with GGO, which appeared one week after inoculation.3. The incidence of nodules was significantly higher in tracheal inoculation group than that in venous inoculation group, and incidence of consolidations and GGO had no significant difference between the two groups.4. Pulmonary candidiasis had three forms including bronchopulmonary form, embolic form and capillary-invasive form, among which embolic form was most common. Bronchopulmonary form only occurred in airway infectious route, and embolic form and capillary-invasive form occurred in the both routes.5. CT findings in pulmonary candidiasis of embolic form had some characteristic features, showing as bilateral multiple nodules of uniform size, with halo sign and without cavitation, which were firstly appeared in the peripheral zone of lung and the rapidly progress to diffuse distribution, pathologically presenting as hemorrhagic and necrotic nodules...