Liesbeth Vanherp

Longitudinal, in vivo assessment of invasive pulmonary aspergillosis in mice by computed tomography and magnetic resonance imaging

Invasive aspergillosis is an emerging threat to public health due to the increasing use of immune suppressive drugs and the emergence of resistance against antifungal drugs. To deal with this threat, research on experimental disease models provides insight into the pathogenesis of infections caused by susceptible and resistant Aspergillus strains and by assessing their response to antifungal drugs. However, standard techniques used to evaluate infection in a preclinical setting are severely limited by their invasive character, thereby precluding evaluation of disease extent and therapy effects in the same animal. To enable non-invasive, longitudinal monitoring of invasive pulmonary aspergillosis in mice, we optimized computed tomography (CT) and magnetic resonance imaging (MRI) techniques for daily follow-up of neutropenic BALB/c mice intranasally infected with A. fumigatus spores. Based on the images, lung parameters (signal intensity, lung tissue volume and total lung volume) were quantified to obtain objective information on disease onset, progression and extent for each animal individually. Fungal lung lesions present in infected animals were successfully visualized and quantified by both CT and MRI. By using an advanced MR pulse sequence with ultrashort echo times, pathological changes within the infected lung became visually and quantitatively detectable at earlier disease stages, thereby providing valuable information on disease onset and progression with high sensitivity. In conclusion, these non-invasive imaging techniques prove to be valuable tools for the longitudinal evaluation of dynamic disease-related changes and differences in disease severity in individual animals that might be readily applied for rapid and cost-efficient drug screening in preclinical models in vivo.

Bronchoscopic fibered confocal fluorescence microscopy for longitudinal in vivo assessment of pulmonary fungal infections in free-breathing mice

Respiratory diseases, such as pulmonary infections, are an important cause of morbidity and mortality worldwide. Preclinical studies often require invasive techniques to evaluate the extent of infection. Fibered confocal fluorescence microscopy (FCFM) is an emerging optical imaging technique that allows for real-time detection of fluorescently labeled cells within live animals, thereby bridging the gap between in vivo whole-body imaging methods and traditional histological examinations. Previously, the use of FCFM in preclinical lung research was limited to endpoint observations due to the invasive procedures required to access lungs. Here, we introduce a bronchoscopic FCFM approach that enabled in vivo visualization and morphological characterisation of fungal cells within lungs of mice suffering from pulmonary Aspergillus or Cryptococcus infections. The minimally invasive character of this approach allowed longitudinal monitoring of infection in free-breathing animals, thereby providing both visual and quantitative information on infection progression. Both the sensitivity and specificity of this technique were high during advanced stages of infection, allowing clear distinction between infected and non-infected animals. In conclusion, our study demonstrates the potential of this novel bronchoscopic FCFM approach to study pulmonary diseases, which can lead to novel insights in disease pathogenesis by allowing longitudinal in vivo microscopic examinations of the lungs.

A multimodal imaging approach enables in vivo assessment of antifungal treatment in a mouse model of invasive pulmonary aspergillosis

ABSTRACT Aspergillus fumigatus causes life-threatening lung infections in immunocompromised patients. Mouse models are extensively used in research to assess the in vivo efficacies of antifungals. In recent years, there has been an increasing interest in the use of noninvasive imaging techniques to evaluate experimental infections. However, single imaging modalities have limitations concerning the type of information they can provide. In this study, magnetic resonance imaging and bioluminescence imaging were combined to obtain longitudinal information on the extent of developing lesions and fungal load in a leukopenic mouse model of invasive pulmonary aspergillosis (IPA). This multimodal imaging approach was used to assess changes occurring within lungs of infected mice receiving voriconazole treatment starting at different time points after infection. The results showed that IPA development depends on the inoculum size used to infect animals and that disease can be successfully prevented or treated by initiating intervention during early stages of infection. Furthermore, we demonstrated that a reduction in fungal load is not necessarily associated with the disappearance of lesions on anatomical lung images, especially when antifungal treatment coincides with immune recovery. In conclusion, multimodal imaging allows an investigation of different aspects of disease progression or recovery by providing complementary information on dynamic processes, which are highly useful for assessing the efficacy of (novel) therapeutic compounds in a time- and labor-efficient manner.