1. Methods
Preparation of Drug and Inoculum:
• Drug A vials were 3 mL: 2.5 mL Drug A at 150 mg/mL in 80% propylene glycol/20% ethanol.
• Stored at 2-8 degrees Celsius.
• Diluted with 5% glucose in water (DW5) to 1.2 mg/mL on each day of dosing.
• Aspergillosis fumigatus clinical isolate Af293 was used to infect the mice.
• This isolate had successfully infected lab animals in past studies done by our lab.
• It was recovered from lung tissue at autopsy from a patient with fatal pulmonary aspergillosis.
• Prior to inoculation, an aliquot of Af293 stock was plated onto potato dextrose agar (PDA) and
allowed to germinate in a humidified incubator for 10 days at 37 degrees Celsius.
• On day of infection, PDA surfaces were flooded with surfactant polysorbate 80 (Tween 80) to
make picking up inoculum with disposable plastic loop easier.
• The conidial suspension was concentrated by high speed centrifugation and then diluted from
1:1000 to 1:10,000. The number of conidia was measured by hemocytometer.
• This inoculum’s viability was tested by plating 3 serial dilutions (1:100) in triplicate, and counting
the colony-forming units after one night of incubation at 37 degrees Celsius.
Immunosuppression and Antibacterial Prophylaxis:
• Aspergillosis fumigatus doesn’t usually induce morbidity in organisms with a healthy immune system.
The mice we were using had to be immunosuppressed for the infection to take place.
• We used a neutropenic regimen consisting of cyclophosphamide and cortisone acetate to do this.
• With their immune systems suppressed, the mice were susceptible to many types of bacteria. To prevent
superinfection, we gave an antibiotic called enrofloxacin to the mice at 50 ppm in their drinking water.
Aerosol Inoculation:
• ~25 gram male ICR mice were placed in an acrylic inhalation chamber that was connected to a nebulizer
containing 6 mL of the inoculum and a tank of compressed air.
• Air was run through the nebulizer at 100kPa for 15 minutes initially. Then, the nebulizer was
loaded with another 6 mL of conidia suspension and aerosolized with 100kPa for 30 minutes.
• After aerosolized inoculation, the mice remained in the chamber for 1 hour of exposure time.
• After exposure, 5 mice were randomly selected, weighted, sacrificed, and had their lungs
aseptically removed. The lungs were weighted, placed in saline with chloramphenicol (400 μg/mL)
and gentamicin (25 μg/mL), and homogenized into serial dilutions from 1:10 to 1:100. 100 μL of
each dilution was then plated onto PDA plates in duplicate. These plates were incubated for 24
hours at 37 degrees Celsius. We then counted the colony forming units to see if infection had, in
fact, taken place in our mice.
Antifungal Therapy:
• Therapy began 24 hours after infection and lasted through day 7 post-infection.
• Drug A was administered subcutaneously, and Posaconazole, our positive control, by oral gavage.
• Refer to the table below to see the number of mice in each treatment group:
Literature Cited
1.Saito, K. Nijima, A. Kamite, E. Watanabe, M.
Bisphenol A and estrone induced developmental
effects in early chick embryos. Environmental
Toxicology 27 (1). January 2012.
2.What is BPA? Is it Harmful? Medical News Today. 4
April 2011. Accessed 6 April 2014.
<http://www.medicalnewstoday.com/articles/221205.ph
p>
3.Chick staging
source<http://msucares.com/poultry/reproductions/poul
try_chicks_embryo.html>
• Survival Treatment Arm:
• Antifungal therapy was continued throughout day 7 post-infection; mice were
monitored off therapy until day 12 and euthanized when they displayed more than
one of the following characteristics of morbidity: ruffled/matted fur, hunched
posture, weight loss (>20%), hypothermia (cool to touch), hypo-/hyperventilation,
and inability to eat or drink.
• Fungal Burden Arm:
• Mice were administered Drug A until their assigned day of termination (D5 or D8),
at which point their lungs were aseptically extracted, massed, homogenized, plated,
incubated, and later inspected to see if any Aspergillosis could be found.
Results
Conclusions
• Drug A does have mechanisms for lowering the amount
of fungal organisms within the host body of a mouse.
• It seemed, however, to be lethal to this model organism.
• The deaths of mice treated with our positive control,
Posaconazole, brings into question the possibility of an
experimental error.
• Posaconazole is a drug that has proven to be effective
against invasive pulmonary aspergillosis; it is used in
humans regularly. It is therefore odd to see the mice
treated with it dying, as they are expected to recover.
• The clearest explanation for our results would be that
our method of treatment somehow killed the mice, but
this doesn’t seem plausible. The mice, before dying, had
shown clear signs of morbidity that are commonly
associated with invasive pulmonary aspergillosis.
Guerrieri, Maximo1,3*, Vargas, Christian2,3, Najvar, Laura 3,4, Jaramillo, Rosie3,4, Olivo, Marcos3, Weiderhold, Nathan P.3 and Patterson, Thomas3,4
1Davidson College, 2Trinity University, 3The University of Texas Health Science Center at San Antonio, and 4South Texas Veterans Healthcare System, San Antonio, TX, USA
Future Steps
• This study will be repeated again. Our results clearly
point towards an experimental error. We cannot see any
fault in our methods that would explain the nature of our
results. Perhaps, a piece of our equipment was faulty.
• Performing the study over again, with new equipment
and mice, may lead to results that relate more directly to
the effectiveness of Drug A on treating invasive
pulmonary aspergillosis.
Acknowledgements
Many thanks to the Davidson Research Network and the work of
Dr. Spencer Redding, Dr. Verna Case, and Linda Shoaf. Also
thanks to Dr. Thomas Patterson’s medical mycology lab
including Dr. Nathan Wiederhold, Dr. Laura Najvar, Rosie
Jaramillo, Marcos Olivo, and Christian Vargas.
Investigating the Effectiveness of a Novel Antifungal Agent Using Murine
Models of Invasive Pulmonary Aspergillosis
Literature Cited
1. Neofytos D, Horn D, Anaisse E, Steinbach W, Olyaei A, Fishman
J, Pfaller M, Chang C, Webster K, Marr K. 2009. Epidemiology
and outcome of invasive fungal infection in adult hematopoietic
stem cell transplant recipients: analysis of Multicenter Prospective
Antifungal Therapy (PATH) Alliance registry. Clin Infect Dis 48:265-
273.
2. Lin SJ, Schranz J, Teutsch SM. 2001. Aspergillosis case-fatility
rate: systematic review of the literature. Clin Infect Dis 32:358-366.
3. Verweij PE, Chowdhary A, Melchers WJ, Meis JF. 2016. Azole
Resistance in Aspergillosis funigatus: Can We Retain the Clinical
Use of Mold-Active Antifungal Azoles? Clin Infect Dis 62:362-368
4. Wiederhold NP, Gil VG, Gutierrez F, Lindner JR, Albataineh MT,
McCarthy DI, Sanders C, Fan H, Fothergill AW, Sutton DA. 2016.
First Detection of TR34 L98H and TR46 Y121F T289A Cyp51
Mutations in Aspergillus funigatus Isolates in the United States. J
Clin Microbiol 54:168-171.
n=19 n=19
n=15
n=15
n=13
n=11 n=14 n=14
Day14
0.5 mm0.5 mm
Day 1
Abstract
Aspergillosis is a major cause of invasive fungal infection that leads to morbidity and
mortality (1, 2). Success rates of present drugs are sub-optimal and concerns
regarding toxicity and drug-to-drug interactions do exist. Additionally, strains of azole-
resistant Aspergillosis fumigatus isolates have been found across the world (3,4).
Azoles are the only orally available agents that treat aspergillosis. This has led
scientists to create and test new drugs. Our lab performed in vivo drug testing in a
murine model (ICR mice) for a certain drug that will be referred to as “drug A” for the
sake of confidentiality. We divided our mice into 7 experimental groups: vehicle
controls; drug dosages of 2, 4, and 8 mg/kg; 20 mg/kg of our positive control,
Posaconazole; sacrifice group 1-hour post-infection; and 5 uninfected control mice.
We divided these groups further into survival (to observe mortality rates) and fungal
burden groups (mice were euthanized on either day 5 or 8 to observe characteristics
of morbidity). Our results showed that the drug did reduce CFU counts of the fungus
with higher dosages, but also increased the mortality of the mice compared to the
vehicle control. This suggests that the drug itself may be lethal.
Treatment Groups Survival Fungal Burden
Placebo Controls (D5W) 10 15 (5 burdened on D5, 10 on D8)
Drug A 2 mg/kg SC BID 10 15 (5 burdened on D5, 10 on D8)
Drug A 4 mg/kg SC BID 10 15 (5 burdened on D5, 10 on D8)
Drug A 8 mg/kg SC BID 10 15 (5 burdened on D5, 10 on D8)
Posaconazole 20 mg/kg PO BID 10 15 (5 burdened on D5, 10 on D8)
1 hour SAC 5 0
Uninfected Immunosuppressed Controls 5 0
Total Mice 60 75
100
101
102
103
104
105
106
CFU/gLungTissue
Lung Fungal Burden Day 5
Control Drug A 2 mg/kg Drug A 4 mg/kg Drug A 8 mg/kg POS 20 mg/kg
BID BID BID BID BID
C
ontrolvehicle
BID
D
rug
A
2
m
g/kg
BID
D
rug
A
4
m
g/kg
BID
D
rug
A
8
m
g/kg
BID
PO
S
20
m
g/kg
BID
1
hrSAC
U
ninf.C
ontrol
100
101
102
103
104
105
106
Lung Fungal Burden Day 8
CFU/gLungTissue
Fig.1 CFU counts of aspergillosis after days 5 and 8 of treatment of various
dosages of drug. Higher dosage led to lower amount of Aspergillosis; highest
dose had the same reduction in CFU counts as our positive control,
Posaconazole.
Results (cont.)
C
ontrolvehicle
BID
D
rug
A
2
m
g/kg
BID
D
rug
A
4
m
g/kg
BID
D
rug
A
8
m
g/kg
BID
PO
S
20
m
g/kg
BID
100
101
102
103
104
105
106
Lung Fungal Burden Survival Group
CFU/gLungTissue
Fig. 2 CFU counts of aspergillosis from survival treatment arm.
Mice were allowed to live for as long as they could, but all were
termed at the end of the experiment. Higher dosage led to lower
amount of Aspergillosis; highest dose had same reduction in CFU
counts as our positive control, Posaconazole.
C
ontrolvehicle
BID
D
rug
A
2
m
g/kg
BID
D
rug
A
4
m
g/kg
BID
D
rug
A
8
m
g/kg
BID
PO
S
20
m
g/kg
BID
1
hrSAC
U
ninfected
C
ontrol
100
101
102
103
104
105
106
Lung Fungal Burden Cumulative
CFU/gLungTissue
Fig. 3 CFU counts of aspergillosis from both treatment arms, fungal burden and survival. Overall,
higher dosage led to lower amount of Aspergillosis; highest dose had the same reduction in CFU
counts as our positive control, Posaconazole.
Fig. 4 Survival rates of all mice in survival treatment arm
(separated by treatment group) throughout entire
experiment. Mice treated with Drug A all died despite
reduction in CFU counts of Aspergillosis. Posaconazole
had a similar effect. Uninfected mice all survived, mice
who were infected but not treated survived at 30%.