Liesbeth Vanherp1, Kristof Govaerts1, Amy Hillen2, Jennifer Poelmans3, Katrien Lagrou4, Greetje Vande Velde1, and Uwe Himmelreich1
1Biomedical MRI, University of Leuven, Leuven, Belgium, 2Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden, 3Janssen Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium, 4Laboratory of Clinical Bacteriology and Mycology, University of Leuven, Leuven, Belgium
Synopsis
Multi-parametric MRI was correlated with in vivo Fibred Confocal Fluorescence Microscopy and histology in two preclinical models of cryptococcosis. Increased ADCs and T2 relaxation times were linked to differences in capsule size and
associated fungal density in brain lesions caused by the two pathogenic fungi Cryptococcus neoformans and C. gattii. This provides not only a non-invasive means to assess one of the most important virulence factors of Cryptococci in preclinical research but may also affect patient management by providing a method for differential diagnosis.
INTRODUCTION
Cryptococcosis, which
is caused by the opportunistic pathogens Cryptococcus
neoformans and C. gattii, is the
most common fungal brain infection [1]. Fungal infections of the central
nervous system are associated with high mortality and morbidity. Infections mainly
occur in immunocompromised patients, but can also affect immunocompetent people
[2]. One of the most important virulence factors of cryptococci is their
polysaccharide capsule, one of the most studied traits in these model organisms [3, 4]. Current methods to assess the capsule size are limited
to ex vivo analysis, thereby
precluding longitudinal analysis of changes in this dynamic structure. We
hypothesize that differences in capsule size can affect the packing of cells
inside a lesion and investigated the fungal cell density and size of cryptococcal
brain lesions in living mice using intravital fibered confocal fluorescence
microscopy (FCFM) and multi-parametric MRI.
Hereby, the model strains C. neoformans H99
(CN) and C. gattii R265
(CG) were used to assess the differences in both species and potentially
provide a basis for differential diagnosis.MATERIAL AND METHODS
Unilateral focal brain lesions were induced by
stereotactic injection of 10
4 GFP-expressing cryptococci [5] (CG R265, n=6
and CN H99, n=5) in the striatum of female Balb/C mice. MR images were acquired on day 9 post infection
using a 9.4/20 Biospec small animal MR system (20cm
horizontal bore with actively
shielded gradients of up to 600mT m
-1, Bruker Biospin, Ettlingen, Germany) and a 7 cm linearly polarized
resonator for transmission and an actively-decoupled mouse brain surface coil
for receiving (Rapid Biomedical, Rimpar, Germany).
The following MRI protocols were acquired:
- T2-weighted
anatomical MRI (TR/ TE = 4200/ 36.2 ms, rare factor 8, slice thickness 0.5mm, in plane resolution 0.1mm
- Diffusion weighted MRI, b-values
up to 1561 s mm-2, TR/ TE = 3000/ 27 ms
- T1-maps using RARE-VTR with TR=272,
350, 750, 1500 and 5000ms
- T2-maps using multi-slice-multi-echo
MRI with 16 TE increments of 10ms, ranging from 10 to 160ms, TR = 2000ms
FCFM was performed on day 10 after infection using a Cellvizio
instrument (Mauna Kea), operating at 488nm. A fibreoptic probe S-300 was
mounted on a stereotactic frame and inserted into the brain lesion.
After sacrificing the animals, colony-forming unit
(CFU) counting on brain homogenates was performed and the size of the fungal
cells and their surrounding capsule was measured after india ink staining using
a microscope with digital camera (Leica DM LS2 with ICC50W). Analysis was
performed using the tool described by Dragotakes et al. [6].
RESULTS
Quantitative
multi-parametric MRI
MRI results are summarized in Figure 1. Lesions
were characterized by higher apparent diffusion coefficients (ADC), T1 and T2 relaxation times
than in the contralateral (normal) hemisphere. The ADC and T2 relaxation times
were significantly higher in CG lesions than in CN lesions, without differences
in the T1
relaxation
time. Clear distinction between CG and CN infection was possible.
In vivo Fibred Confocal
Fluorescence Microscopy
FCFM results are summarized in Figure 2.
FCFM showed that the cryptococcal cell density was higher in lesions caused by
CN H99 than by CG R265. The number of cells (CFU) per mm³ of lesion material
was significantly higher in CN lesions, confirming more densely packed cells in
CN lesions.
Ex vivo confirmation
India ink staining of the homogenized
brain tissue showed that CG R265 cells are in general larger
than CN H99 cells, with a thicker
capsule around the cell and a larger cell body size, leading to a lower cell
density compared to CN (Figure 3).DISCUSSION
The higher ADC and T2 relaxation time in
CG lesions compared to infections caused by CN can be explained by the larger
fluid-filled intercellular spaces and higher amount of the highly hydrated
capsular material in CG. This has been summarized in the model shown in Figure 4. The lower fungal density in CG R265 lesions was confirmed both by the FCFM
images and by calculating the number of CFUs per lesion volume. India ink
staining confirmed that CG cells were in general larger than CN cells, with a
thicker capsule around the cell and larger cell body [3, 4], which explains the
lower cell density compared to CN. While the larger capsule size for CG
compared to CN confirms previous ex vivo studies [3, 4], more clinical strains are
required to generalize our findings.
We hereby propose a method that allows in vivo measurement of the fungal cell density in cryptococcal
lesions, leading to indirect information about the capsule size in an in vivo setting. We are currently
investigating the potential of FCFM and MRI to provide longitudinal insights in
changes of fungal density throughout the disease progression.CONCLUSIONS
Increased ADCs and T2 relaxation times were
used to distinguish between CG R265 and CN H99 lesions,
which was related to the capsule size
and associated fungal density in the respective lesions. If confirmed by
studies using more clinical strains this multi-parametric MRI approach could be
used to differentiate between CN and CG. As manifestation and treatment of
cryptococcosis caused by CN or CG is different [7], this may impact patient management
in the future.Acknowledgements
We are thankful for financial support
from the European Commission for the Infect-ERA project
CryptoVIEW. LV is an SB PhD fellow at Research
Foundation Flanders (FWO).
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