Diagnostic Performance of Contrast Enhanced Pulmonary Computed Tomography Angiography for the Detection of Angioinvasive Pulmonary Aspergillosis in Immunocompromised Patients

Invasive pulmonary aspergillosis (IPA) is one of the major complications in immunocompromised patients. The mainstay of diagnostic imaging is non-enhanced chest-computed-tomography (CT), for which various non-specific signs for IPA have been described. However, contrast-enhanced CT pulmonary angiography (CTPA) has shown promising results, as the vessel occlusion sign (VOS) seems to be more sensitive and specific for IPA in hematologic patients. The aim of this study was to evaluate the diagnostic accuracy of CTPA in a larger cohort including non-hematologic immunocompromised patients. CTPA studies of 78 consecutive immunocompromised patients with proven/probable IPA were analyzed. 45 immunocompromised patients without IPA served as a control group. Diagnostic performance of CTPA-detected VOS and of radiological signs that do not require contrast-media were analyzed. Of 12 evaluable radiological signs, five were found to be significantly associated with IPA. The VOS showed the highest diagnostic performance with a sensitivity of 0.94, specificity of 0.71 and a diagnostic odds-ratio of 36.8. Regression analysis revealed the two strongest independent radiological predictors for IPA to be the VOS and the halo sign. The VOS is highly suggestive for IPA in immunocompromised patients in general. Thus, contrast-enhanced CTPA superior over non-contrast_enhanced chest-CT in patients with suspected IPA.


Patients and Methods
Patients. This retrospective single-centre study was approved by our institutional review board and complies with both the Declaration of Helsinki and the Health Insurance Portability and Accountability Act (HIPAA). Due to the retrospective nature of the study protocol, the local ethical committee waived the need for written informed consent.
We retrospectively analysed patients' electronic records and hospital charts from 1999 to 2014 and identified 455 immunocompromised patients with proven/probable IPA based on the 2008 EORTC/MSG consensus definitions. Of these 455 patients, 78 received a CTPA study for the diagnostic work-up during an infectious episode of suspected IPA and were therefore included in this study. Patient characteristics are summarized in Table 1.
Forty-five equally immunocompromised patients with pulmonary infiltrates but no evidence of IPA based on the EORTC/MSG criteria served as a control group (NoIPA group). In the majority of patients, extended efforts were taken to identify the infectious organism, e.g. by performing bronchoscopy with bronchoalveolar lavage (BAL).

Definitions for Classification.
Immunocompromised patients were classified based on the 2008 EORTC/ MSG consensus criteria 3 . ICU patients who did not meet the criteria for immunosuppression by the EORTC/ MSG but had evidence of Aspergillus involvement were classified according to the criteria proposed by Blot et al., which have been shown to perform better in identifying IPA in ICU patients 4 . Radiological examinations. CTPA studies were performed either on a 16-slice MDCT system (SOMATOM Emotion, Siemens Healthineers, Forchheim, Germany) or on a 1 st generation 2 × 32 slice dual-source CT system (SOMATOM Definition, Siemens Healthineers, Forchheim, Germany). The contrast injection protocol included the injection of 80 ml of Iomeprol (Imeron 400, Bracco, Milan, Italy) with a flow-rate of 4 ml/s via an antecubital vein followed by a 40 ml saline chaser using the same injection rate. The bolus tracking method was used to determine the scan start. The scan automatically began 5 s after a threshold of 100 HU was reached within a region of interest placed within the pulmonary trunk, with scanning performed in a cranio-caudal direction.
Images were re-analysed for this study in a consensus reading by two chest radiologists (CH, TH) blinded to the patients' diagnoses, clinical courses, and complications. The definition of the observed patterns followed the glossary of terms for thoracic imaging proposed by the Fleischner Society 9 . The following radiological patterns were evaluated: According to Sonnet et al., vascular occlusion was defined as a vessel interruption at the border of a focal lesion without extension of the vessel into or out of the lesion 7 . The presence of the VOS was only evaluated in lesions with a diameter ≥ 10 mm (or >12 mm in the peripheral lung).
Timing of CTPA. As radiological patterns of IPA have been shown to change over the course of the infection and the halo sign being an early indicator of IPA 10 , the time of CTPA relative to the start of the infectious episode (as defined by the beginning of the increase of systemic inflammation markers [C-reactive protein or procalcitonine]) was evaluated in a subgroup of patients.
Evaluation of renal function. Baseline and follow-up serum creatinine values were recorded to evaluate potential nephrotoxicity caused by contrast agent administration. Significant contrast-agent mediated nephrotoxicity was defined according to Stacul et al. 11 . Briefly, contrast medium-induced nephropathy (CIN) was defined as an increase in serum creatinine by ≥ 25% within 3 days after contrast agent administration in the absence of plausible alternative etiologies. Statistical analysis. Diagnostic performance of CTPA was calculated by comparing all proven/probable IPA patients (n = 78) with the No-IPA (n = 45) control population. Sensitivity analysis was performed; odds ratios and 95%-confidence interval of these ratios were calculated. For discrete variables Fishers' exact test and the χ²-test were used, as indicated.
For the logistic regression analysis, the leading response (dichotomous) variable was proven/probable IPA using a binary logit model with Fisher's scoring as an optimization technique. The entry probability into the logistic regression model was set to 0.45 and the probability of stay was 0.55. Model building was started with all 12 diagnostic imaging variables. Goodness of fit was tested by deviance, Pearsons' test, and the Hosmer-Lemeshow test. Level of significance was set to 0.05. Statistical software SAS 9.4 (SAS Institute Inc., Cary, NC, USA) were used for descriptive statistics, sensitivity analysis, and logistic regression and for the latter also LogXact software (Cytel Studio, Version 9.00, Cytel Inc. Pune, USA).

Results
Patient characteristics. Characteristics of the 78 immunocompromised patients with proven/probable IPA and an available CTPA study are summarized in Table 1. Among all patients with proven/probable IPA, 41 had an underlying hematological disease, whereas the remaining 37 were immunocompromised due to other conditions, such as solid-organ-transplantation, chronic obstructive pulmonary disease (COPD) with intensive systemic steroid treatment, or autoimmune diseases such as granulomatosis with polyangiitis (formerly Wegener's granulomatosis). All of these patients met the EORTC or Blot criteria based on intensity of underlying or iatrogenic immunosuppression. Based on microbiological results obtained from BAL, sputum culture, respiratory specimen or blood culture, 42/45 patients in the immunocompromised control cohort were found to have an infectious disease other than aspergillosis or mucormycosis; no etiological pathogen could be identified in the remaining three patients. Twenty-two percent of patients had been treated with antifungal therapy (AFT) prior to performance of CTPA defined as receiving > 1 daily dosage mould-active AFT.
Altogether, five radiological patterns were associated with IPA in a statistically significant manner: The halo sign, the VOS, internal low attenuation, the air-crescent sign and the nodule pattern (all p < 0.05). Detailed diagnostic performance of the different radiological signs is displayed in Table 2.
Frequency of VOS or halo sign in hematological and non-hematological conditions. We performed a subanalysis to evaluate whether the prevalence of VOS differs in immunocompromised patients with hematological disorders versus those without. VOS was seen in 33/41 (80%) patients with hematologic disorders and in 28/37 (76%) patients without hematologic disorders (no statistical difference; p > 0.6). Figures 1 and 2 display examples of VOS in immunocompromised patients with hematologic and non-hematologic conditions.
There was also no significant statistical difference in the prevalence of the halo sign between groups, seen in 32/41 (78%) patients with hematologic disorders and 24/37 (65%) patients without hematological disorders, although the difference was more pronounced (p > 0.2).
Four patients in the control group showed a positive VOS: One NoIPA patient suffered from COPD with chronic systemic steroid treatment; however, steroid dose was not high enough to meet EORTC host criteria. In addition to the observed VOS, the patient showed infiltrate patterns typical for IPA, a positive halo sign, and positive blood Galactomannan (GM) as mycological evidence. The second VOS-positive NoIPA case was an ICU patient with multiorgan failure after polytrauma, positive GM in blood, and pulmonary embolism, possibly explaining the observed VOS. The third VOS-positive NoIPA patient also suffered from COPD and showed typical infiltrate patterns (including halo sign) with positive GM from blood and BAL. Due to insufficient host criteria; however, this patient was classified as NoIPA. The fourth VOS-positive NoIPA patient suffered from abscessing pneumonia and had a positive halo sign and positive blood GM. However, immunocompromising conditions were not strong enough according to EORTC/Blot criteria; accordingly, the patient was classified as NoIPA.
When comparing the diagnostic performance of the different radiological patterns, the VOS sign was found to be the strongest of all evaluated radiological signs in the detection of IPA. Table 2 summarizes the diagnostic performance of the different radiological signs.

Diagnostic performance of VOS and halo sign relative to the start of the infection. Median time
to CT was 7 days (range 0-41 days) for patients with proven/probable IPA wheareas it was 6 days (range 0-55 days) for patients with NoIPA. Statistical comparison between both proven/probable IPA vs. No IPA patients showed no significant difference between both groups (p > 0.92; Mann-Whitney-U test). As the halo sign has been shown to be an early indicator of IPA, subanalysis of patients who received the CT-scan+/−2 days relative to start of the infectious episode was performed: In these patients positivity for the halo sign was found in 9/17 proven/probable IPA patients (53%), while the VOS was positive in 15/17 of these patients (93%). Statistical analysis found the VOS to be significantly more frequent compared to the halo sign (p < 0.028; Mann-Whitney-U test).

Influence of Antifungal treatment on VOS frequency. The vessel occlusion sign was detected in 86%
of patients without mould-active agents prior to CTPA while positivity of VOS could be demonstrated for 73% of patients receiving AFT prior to performance of CTPA. This difference was not found to be statistically significant (p > 0.31; Mann-Whitney-U test). Four patients had received more than one mould-active antifungal agent prior to CTPA (two patients had liposomal Amphotericin B and Caspofungin, one patient had caspofungin and  Statistical Evaluation and logistic regression model. Of the twelve evaluated radiological patterns, five radiological signs were found to be significantly associated with IPA: The VOS, the halo sign, the air crescent sign, the internal low attenuation sign, and the nodular configuration. Each showed a capacity of dichotomization between IPA and NoIPA patients with a p-value of <0.05. The vessel occlusion sign (VOS), with the superior diagnostic odds ratio, served as the best discriminator between IPA and NoIPA.
Stepwise logistic regression model. A logistic regression analysis was performed to check for independent association of these radiological signs, which often occur concurrently in the clinical setting. A stable model was built using the following seven variables which were entered into a stepwise procedure: VOS, halo sign, crazy paving, nodules, infarct shaped infiltrate, cavern and the air-crescent sign. Goodness of fit test showed a p-value of 0.77 in the the Hosmer-Lemeshow test, indicating that the model fits. A sum score model (adding up all 7 dichotomous variables) was built and seven observations were read. Goodness of fit test (deviance and Pearsons' test) was given.
The Receiver Operating Characteristic (ROC) analysis using these seven radiological signs is displayed in Fig. 3. The ROC had a steep initial increase and an area under the curve (AUC) of 0.805, suggesting a good positive diagnostic value. Removing the VOS from the model lead to a distinct decrease of AUC from 0.805 to 0.705, underlining the diagnostic placevalue of the VOS.
The logistic regression analysis revealed VOS and the halo sign as the two strongest independent radiological predictors for proven/probable IPA, with the VOS showing the most significant score (Chi-Square score 52.93; p < 0.0001, χ²-test).
Safety of CTPA regarding nephrotoxicity. Median serum creatinine was 0.90 mg/dl (range 0.29-7.12 mg/dl) before CTPA (baseline) and 0.95 mg/dl (range 0.34-6.2 mg/dl) 48-72 hours after contrast agent exposure. Only two patients experienced an increase in serum creatinine of more than 25%, both however staying within the normal range, indicating a very tolerable rate of acute kidney injury according to published CIN criteria 11 .

Discussion
Our study demonstrates that VOS as observed on CTPA examinations is superior to classic CT signs observed in non-contrast enhanced studies to diagnose invasive pulmonary aspergillosis in immunocompromised patients. This result held true regardless of the hematological or non-hematological origin of their condition and timing of the CT scan. As had been reported previously, we could not detect a significant influence of underlying antifungal therapy/prophylaxis on VOS performance. In addition, the use of intravenously injected contrast medium did not lead to nephrotoxicity in this patient cohort. This finding is in accordance to recent studies that questioned the harmfulness of intravenously injected contrast material even in patients with known kidney disease 12 .
IPA is a frequent but difficult diagnosis in severely immunocompromised patients 13 . Only histological examination and/or positive culture results from sterile sites provide definitive proof of IPA according to the latest EORTC/MSG consensus definitions 3 . Microbiological tests such as cultures or even biomarkers are often negative, especially under standard prophylactic or empiric antifungal medication 14,15 . While this is often implemented as a routine clinical strategy, basing the diagnosis of IPA on non-contrast enhanced chest CT criteria alone is highly questionable. Only 50% of patients diagnosed with IPA by conventional CT criteria were found to truly harbor invasive fungal disease in microbiological examinations after surgical resection 6 . Other pulmonary infectious diseases can show similar radiological patterns 16 . At the same time, the hallmark halo sign 5, 17 is of only short duration and thus easy to miss 10 , leading to a real-life sensitivity of about 0.8 in other studies and our own data 5 so a considerable proportion of patients with IPA will go undiagnosed. Newer radiological techniques such as positron emitting tomography 18 or magnetic resonance imaging 19 are currently under evaluation for this diagnosis, but these tend to be less available and more laborious, time-consuming, and expensive.
By contrast, CTPA would provide an easily available improvement of the currently recommended imaging standard, and its non-invasiveness as opposed to bronchoscopy represents a clear advantage in immunocompromised patients who often are in critical clinical condition and at increased bleeding risk, prohibiting invasive diagnostic procedures. Moreover, as early diagnosis and early initiation of therapy improve survival in IPA patients 20 , the constant availability of CTPA in most institutions allows for fast and direct therapeutic decision-making, whereas bronchoscopy and subsequent pathological or microbiological examination as well as laboratory diagnostics of biomarkers can delay diagnosis for several days 21 .
As different radiological patterns often appear concurrently, a logistic regression analysis to further elucidate the independent diagnostic value of each radiological sign was performed and revealed that VOS independently was superior to the halo sign.
These findings of superior diagnostic performance of the contrast-enhanced VOS are in accordance with those of Stanzani et al. who initially proposed the use of CTPA in immunocompromised patients with hematologic disorders and suspected IPA 22 .
In addition to the former study, however, we broadened our cohort and for the first time included immunocompromised patients with conditions other than hematologic diseases.
It has been proposed that the prevalence of radiological patterns might be different depending on the disorder causing the reduced immunocompetence. In particular, hematologic malignancies were rather supposed to be associated with the halo sign compared to other conditions 23 . We did not find a significant difference between these groups for either VOS or the halo sign, although the difference in sensitivity between VOS and the halo sign was more pronounced in the non-hematological group. Furthermore we compared the diagnostic performance of VOS in neutropenic and non-neutropenic patients and could clearly underline its clinical usefulness even in non-neutropenic patients.
A concern in using iodine-based contrast media, especially in critically ill patients, is the risk of acute nephrotoxicity. In this study however, only few patients experienced CIN, and these even stayed within the normal kreatinine range, indicating that CTPA is safe in this patient population with, however, only moderately increased baseline creatinine prior to CTPA. This observation contrasted a number of factors in these patients, such as these patients suffering from or being prone to acute infections, being immunocompromised, some having received allogeneic transplants and being treated with cyclosporine, all of which increase the risk for renal failure [29;30]. These results are supported by previous findings questioning the causation of acute kidney injury by intravenous contrast media administration in a pivotal analysis encompassing more than 20.000 patients which found no excess risk of CIN, dialysis, or death among patients with comorbidities previously reported to predispose to nephrotoxicity 12 .
Whether the phenomenon of vessel occlusion is specific for aspergillosis (the most frequent IMD) or a sign of invasive pulmonary mold disease in general, cannot currently be revealed. Among the five proven cases according to EORTC/MSG definition, Stanzani's cohort included one case of proven mucormycosis who was also positive for VOS. This side-finding supports preclinical data suggesting that Mucor spp. may also have angioinvasive properties 24 and warrants studies similar to ours at centers with higher rates of mucormycosis. Although even rarer invasive pulmonary mold infections caused by Zygomycetes or Fusarium spp. may become more frequent in the future 25 , and despite a broad range in incidences based on local flora 26 , Aspergillus spp. still accounts for more than 90% of invasive human mold infections 27 , substantiating the finding of our study. In either case, the results of this study should apply to the majority of immunocompromised patients with suspected invasive pulmonary mold disease, regardless of whether the diagnostic value of CTPA is pathogen-or species-specific.
Our study has several potential limitations that have to be acknowledged. First, due to the retrospective monocentric design, a selection bias is possible. The prolonged timeframe of the study -with possible changes in patient management and procedures -might have indirectly affected the results. Although CTPA techniques could have varied during the observation period, it appears however, difficult to conceive a direction of bias due to this variation, furthermore the decision to perform CTPA instead of non-contrast enhanced CT was left at the radiologists discretion, possibly representing a confounder. As we retrospectively screened for patients with IPA, we did not specifically focus on populations with other defined infectious etiologies and therefore cannot rule out that other pathogens also display VOS.
The number of 78 proven/probable IPA patients with available CTPA may seem rather small given the long timeframe of the study; this is explained by current clinical practice, wherein the standard diagnostic recommendation is a non-contrast enhanced chest CT in most guidelines concerning immunocompromised patients 2, 28 . In addition, as the control group had to be equally immunocompromised it is probable that some of these control patients did nonetheless suffer from IPA, but could ultimately, not fulfill the very strict EORTC/MSG or Blot criteria for severe immunosuppression. Indeed, all four VOS-positive patients in the control group did show some form of microbiological evidence of IA (GM in Serum or BAL), but had however also evidence of other infections (invasive Candidiasis, A. baumannii, C. freundii). Thus, the diagnostic performance of VOS in our study might have been (negatively) impacted. Finally, as an inherent limitation of CTPA it should be mentioned that small lesions with a diameter ≤ 10 mm are not adequately evaluable for the presence of the VOS 22 .
Scientific RepoRts | 7: 4483 | DOI:10.1038/s41598-017-04470-6 In conclusion, this study confirmed that detection of VOS by CTPA in immunocompromised patients is highly suggestive of IPA and that this observation is independent of hematologic malignancies as opposed to other immunosuppressing underlying diseases. Thus, CTPA studies are likely to have additional diagnostic value over non-contrast enhanced chest CT in immunocompromised patients with suspected IPA. In addition, our data suggests that CTPA is safe with regard to a possible induction of CIN in these patients.
Therefore, this study encourages clinicians to perform CTPA on an individual basis if IPA in immunocompromised patients is suspected and diagnosis is otherwise uncertain. However, before this procedure can become a general recommendation, it needs to be validated prospectively, preferably in a multicentric trial.