High-Resolution Ultrasound Including Contrast-Enhanced Ultrasound (CEUS) for the Detection of Gas Formation during Aspergillus Fumigatus Infection in Mice

Abstract

Purpose: A. fumigatus infections represent a major threat for patients with a suppressed immune system. Early diagnosis is of importance for a favorable outcome but appears to be difficult due to limited diagnostic procedures. Here we investigated the sensitivity of high-resolution ultrasound (HRU) for the detection of A. fumigatus infection in the liver.

Materials and Methods: BALB/c mice were intravenously infected with A. fumigatus and monitored by HRU, Doppler sonography (CCDS), contrast-enhanced ultrasound (CEUS), and real-time strain color-coded elastography (CCE) using a multi-frequency probe (6 – 15 MHz). Contrast media bolus injection of sulfur-hexafluoride micro-bubbles was applied and digital cine-loops from the arterial phase, as well as the portal venous phase up to the late phase of the whole liver were analyzed. All data were correlated to the histopathological findings.

Results: Using HRU and CEUS, a sonic shadow was detected in all infected animals. All Aspergillus-infected nodes from 3 – 6 mm in the liver showed a shadow with rim enhancement and no intranodal enhancement when using CEUS. A. fumigatus infection was confirmed by CFU assessment and histopathological analysis. Granulomas were not associated with shadowing on B-mode. In contrast, granulomas with a diameter above 5 mm and a higher stiffness in CCE generated particularly an arterial rim enhancement and portal venous washout without contrast media uptake in the late phase. In addition, CEUS was able to define dynamic capillary microvascularization of infected liver areas.

Conclusion: Liver lesions associated with A. fumigatus infection can be detected in mice when combined with CEUS and CCE in vivo.

Zusammenfassung

Ziel: A. fumigatus Infektionen stellen eine große Bedrohung für immunsupprimierte Patienten dar. Eine frühe Diagnose ist dabei entscheidend für den Behandlungserfolg, erweist sich bislang allerdings als schwierig. Ziel dieses Projektes war es, die Sensitivität von hochauflösendem Ultraschall (HRU) zur frühen Diagnose einer Pilzinfektion in der Leber zu ermitteln.

Material und Methode: BALB/c Mäuse wurden intravenös mit A. fumigates infiziert und mittels HRU, farbkodierter Sonografie (CCE), Doppler (CCDS) sowie Kontrast-verstärktem Ultraschall (CEUS) unter Einsatz einer Multi-Frequenz Sonde (6 – 15 MHz) untersucht. Kontrastmittel-Bolus-Injektionen von Schwefelhexafluorid Mikrobläschen wurden eingesetzt und digitale Cine-loops von der frühen arteriellen bis zur späten venösen Phase ausgewertet und mit den histopathologischen Ergebnissen korreliert.

Ergebnisse: Mittels HRU und CEUS konnten sonographische Schatten in der Leber aller infizierten Tiere nachgewiesen werden. Die Kontrastmittel-verstärkte Sonografie zeigte bei allen A. fumigatus infizierten Granulomen ab einer Größe von 3 – 6 mm einen Schallschatten in der Leber mit deutlicher Randverstärkung, aber keine Verstärkung im intra-granulären Bereich. Die A. fumigatus-Infektion wurde mittels pathohistologischer Untersuchung und CFU Bestimmung bestätigt. Nicht infizierte Granulome zeigten keinen Schallschatten, aber ab einer Größe von 5 mm und höherer Gewebeverhärtung in CCE induzierten eine Anreicherung von Kontrastmittel im Randbereich in der arteriellen Phase und portal-venöser Auswaschung in der späten Phase (CEUS). Zusätzlich war CEUS in der Lage, dynamische Mikrovaskularisierung in infizierten Leberarealen darzustellen.

Schlussfolgerung: A. fumigatus-Infektionen lassen sich in der Leber mittels HRU in Kombination mit CEUS und CCE in vivo nachweisen.

Key words

Aspergillus fumigatus - inflammation - real-time strain color-coded elastography (CCE) - liver infection - contrast-enhanced ultrasound (CEUS)

^* Authorship due to equal contribution.

• References

• 1 Segal BH. Aspergillosis. N Engl J Med 2009; 360: 1870-1884
• 2 Saliba F, Delvart V, Ichaï P et al. Outcomes associated with amphotericin B lipid complex (ABLC) prophylaxis in high-risk liver transplant patients. Med Mycol 2013; 51: 155-163
• 3 Briegel J, Forst H, Spill B et al. Risk factors for systemic fungal infections in liver transplant recipients. Eur J Clin Microbiol Infect Dis 1995; 14: 375-382
• 4 Singh N, Arnow PM, Bonham A et al. Invasive aspergillosis in liver transplant recipients in the 1990s. Transplantation 1997; 64: 716-720
• 5 Collins LA, Samore MH, Roberts MS et al. Risk factors for invasive fungal infections complicating orthotopic liver transplantation. J Infect Dis 1994; 170: 644-652
• 6 Brakhage AA. Systemic fungal infections caused by Aspergillus species: epidemiology, infection process and virulence determinants. Curr Drug Targets 2005; 6: 875-886
• 7 Ben-Ami R, Lewis RE, Kontoyiannis DP. Enemy of the (immunosuppressed) state: an update on the pathogenesis of Aspergillus fumigatus infection. Br J Haematol 2010; 150: 406-117
• 8 Speth C, Hagleitner M, Ott HW et al. Aspergillus fumigatus activates thrombocytes by secretion of soluble compounds. J Infect Dis 2013; 207: 823-833
• 9 Ben-Ami R, Lewis RE, Leventakos K et al. Aspergillus fumigatus inhibits angiogenesis through the production of gliotoxin and other secondary metabolites. Blood 2009; 114: 5393-5399
• 10 Willger SD, Puttikamonkul S, Kim KH et al. A sterol-regulatory element binding protein is required for cell polarity, hypoxia adaptation, azole drug resistance, and virulence in Aspergillus fumigatus. PLoS Pathog 2008; 4: e1000200
• 11 Grahl N, Puttikamonkul S, Macdonald JM et al. In vivo hypoxia and a fungal alcohol dehydrogenase influence the pathogenesis of invasive pulmonary aspergillosis. PLoS Pathog 2011; 7: e1002145
• 12 Greis C. Ultrasound contrast agents as markers of vascularity and microcirculation. Clin Hemorheol Microcirc 2009; 43: 1-9
• 13 Leen E, Ceccotti P, Kalogeropoulou C et al. Prospective multicenter trial evaluating a novel method of characterizing focal liver lesions using contrast-enhanced sonography. Am J Roentgenol Am J Roentgenol 2006; 186: 1551-1559
• 14 Quaia E, Calliada F, Bertolotto M et al. Characterization of focal liver lesions with contrast-specific US modes and a sulfur hexafluoride-filled microbubble contrast agent: diagnostic performance and confidence. Radiology 2004; 232: 420-430
• 15 Strobel D, Bernatik T, Blank W et al. Diagnostic accuracy of CEUS in the differential diagnosis of small (≤ 20  mm) and subcentimetric (≤ 10  mm) focal liver lesions in comparison with histology. Results of the DEGUM multicenter trial. Ultraschall in Med 2011; 32: 593-597
• 16 Wilson SR, Burns PN. An algorithm for the diagnosis of focal liver masses using microbubble contrast-enhanced pulse-inversion sonography. Am J Roentgenol Am J Roentgenol 2006; 186: 1401-1412
• 17 Wilson SR, Burns PN. Microbubble-enhanced US in body imaging: what role?. Radiology 2010; 257: 24-39
• 18 Wege AK, Schardt K, Schaefer S et al. High resolution ultrasound including elastography and contrast-enhanced ultrasound (CEUS) for early detection and characterization of liver lesions in the humanized tumor mouse model. Clin Hemorheol Microcirc 2012; 52: 93-106
• 19 Lin SJ, Schranz J, Teutsch SM. Aspergillosis case-fatality rate: systematic review of the literature. Clin Infect Dis 2001; 32: 358-366
• 20 Ben-Ami R, Lewis RE, Kontoyiannis DP. Enemy of the (immunosuppressed) state: an update on the pathogenesis of Aspergillus fumigatus infection. Br J Haematol 2010; 150: 406-417
• 21 Bonnett CR, Cornish EJ, Harmsen AG et al. Early neutrophil recruitment and aggregation in the murine lung inhibit germination of Aspergillus fumigatus Conidia. Infect Immun 2006; 74: 6528-6539
• 22 Zarember KA, Sugui JA, Chang YC et al. Human polymorphonuclear leukocytes inhibit Aspergillus fumigatus conidial growth by lactoferrin-mediated iron depletion. J Immunol 2007; 178: 6367-6373
• 23 Kamai Y, Chiang LY, Lopes Bezerra LM et al. Interactions of Aspergillus fumigatus with vascular endothelial cells. Med Mycol 2006; 44: 115-117
• 24 Teutschbein J, Albrecht D, Pötsch M et al. Proteome profiling and functional classification of intracellular proteins from conidia of the human-pathogenic mold Aspergillus fumigatus. J Proteome Res 2010; 9: 3427-3442
• 25 Aimanianda V, Bayry J, Bozza S et al. Surface hydrophobin prevents immune recognition of airborne fungal spores. Nature 2009; 460: 1117-1121
• 26 Carrion Sde J, Leal Jr SM, Ghannoum MA et al. The RodA hydrophobin on Aspergillus fumigatus spores masks dectin-1- and dectin-2-dependent responses and enhances fungal survival in vivo. J Immunol 2013; 191: 2581-2588
• 27 Rødland EK, Ueland T, Pedersen TM et al. Activation of platelets by Aspergillus fumigatus and potential role of platelets in the immunopathogenesis of Aspergillosis. Infect Immun 2010; 78: 1269-1275
• 28 Park SY, Kim SH, Choi SH et al. Clinical and radiological features of invasive pulmonary aspergillosis in transplant recipients and neutropenic patients. Transpl Infect Dis 2010; 12: 309-315
• 29 Muñoz P, Vena A, Cerón I et al. Invasive pulmonary aspergillosis in heart transplant recipients: two radiologic patterns with a different prognosis. J Heart Lung Transplant 2014; 33: 1034-1040
• 30 Franquet T, Müller NL, Giménez A et al. Spectrum of pulmonary aspergillosis: histologic, clinical, and radiologic findings. Radiographics 2001; 21: 825-837
• 31 Ahmad M, Dakshinamurty KV. Emphysematous renal tract disease due to Aspergillus fumigatus. J Assoc Physicians India 2004; 52: 495-497
• 32 Akpinar E, Ayyildiz VA, Petekkaya I et al. Fluid rim sign: a new ultrasonographic sign of soft tissue aspergillosis. Diagn Interv Radiol 2013; 19: 237-239
• 33 Hinson KF, Moon AJ, Plummer NS. Broncho-pulmonary aspergillosis; a review and a report of eight new cases. Thorax 1952; 7: 317-333