BABESIOSIS FINAL PREP
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Transcript of BABESIOSIS FINAL PREP
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Human Babesiosis: The development of a new line of
drugs
Azan Virji
STARS Research Fellow 2015
PI: Professor Ben Mamoun Choukri
Mentor: Lauren Lawres
Department of Infectious Diseases, Yale School of Medicine
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What is Babesiosis?
• Babesia microti
• Northeast & Northern
Midwestern States
• Spread by ticks
http://www.cdc.gov/parasites/images/babesiosis/map_babesiosis_by_county_2013.jpg
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Babesiosis is commonly
misdiagnosed as malaria
https://classconnection.s3.amazonaws.com/915/flashcards/2974915/png/babesia1326269959514-14663CD9F46086C79A4.pnghttp://blogs.cdc.gov/global/2014/02/24/dpdx-15-years-of-strengthening-laboratory-capacity-for-parasitic-disease-
diagnosis/b_microti_vs_p_falciparum-2/
Babesia microti Plasmodium falciparum
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Current therapies against Babesiosis
Vannier, E. et al. (2012, New England Journal of Medicine)
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Recrudescence:
Resistance against current therapies
Treatment
Pereira, M. E. S. et al. (1996, Memórias do Instituto Oswaldo Cruz)
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Addressing the problem
with current therapies
Current therapies are less effective than the new drug Endochin-like Quinolones
(ELQ)
Why are current therapies ineffective?
DNA Sequencing
In vivo study
SCID Mice10 mg/kg by oral gavage
Light Microscope counting
Day 45PCR- Cytochrome b gene
Yale Keck Sequencing Facility
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Three of the four current therapies
are ineffective
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30
% P
aras
item
ia
Days post infection
Giemsa Counts
CONTROL
AZITHROMYCIN
QUININE
CLINDAMYCIN
Treatment
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Atavaquone delays recrudescence
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30 35 40 45 50
% P
aras
item
ia
Days post infection
Giemsa Counts
CONTROL
ATAVAQUONE
Treatment
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Endochin-like Quinolones also
delay recrudescence
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30 35 40 45 50
% P
aras
item
ia
Days post infection
Giemsa Counts
CONTROL
ELQ
Treatment
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ATV + ELQ eliminates
recrudescence
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30 35 40 45 50
% P
aras
item
ia
Days post infection
Giemsa Counts
CONTROL
ELQ + ATV
Treatment
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ATV + ELQ eliminates
recrudescence
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0 5 10 15 20 25 30 35 40 45 50
% P
aras
item
ia
Days post infection
Giemsa Counts
ELQ + ATV
Treatment
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Why does a combination of
ATV and ELQ work better?
CL1
CL2
CL3
CL1
CL2
EL
QV
Sarewicz, M. et al. (2015, jhjournal)
Atavaquone
ELQ
GCT GTTAlanine Valine
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Implications of this research
• Synergy
• Probability that mutation arises is lowered
10-7
ATV/ELQ
10-14
ATV+ELQ
• 2 mutations may lower fitness• Next steps: Dosing, Clinical trials
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Acknowledgements
• Laboratory Of Infectious Diseaseso PI: Professor Ben Mamoun Choukri
o Mentor: Lauren Lawres
o Isaline and Pierre
o Yale School of Medicine
• STARS Summer Research Programo Dr. Moreno, Dr. Nelson and Dr. P And the TAs
o Howard Hughes Medical Institute (HHMI)
o Yale College
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A%
Pa
ra
sit
em
ia
Co
ntr
ol
Qu
inin
e
Clin
dam
ycin
Azit
hro
mycin
Ato
vo
qu
on
e
En
do
ch
in
Ar t
esu
nate
0
2
4
6
8
*
*
*
B
FS
H
3.04%Control
YOYO Quinine
Endochin
Atovoquone
Artesunate Azithromycin
3.99
Clindamycin
3.40
4.5
1.02
1.3
2.8
3.01
Control
FS
C
YOYO-1Figure 1
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Figure 2
% P
ara
sit
em
ia
- V 1 1 0 1 1 0 1 1 0
0
1
2
3
4
5
E L Q 2 7 1 E L Q 3 0 0 E L Q 3 1 6
V
4.66%
ELQ-1
1.50%F
SC
YOYO-1
FS
C
YOYO-1
ELQ-2
1.52%
ELQ-3
1.28%
FS
C
YOYO-1
FS
C
YOYO-1
A
BELQ 1 ELQ 2 ELQ 3
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Figure 3 A B
C D
ELQ 1 ELQ 2
ELQ 3 ELQ 4
0 .0 0
0 .0 5
0 .1 0
2 .5
5 .0
2 5
5 0
7 5
E L Q 2 7 1 (1 0 m g /K g X 7 )
m o n ito r e d b y S m e a r
D a y s p o s t in fe c t io n
% P
ara
sit
em
ia
M o u s e # 1 6
M o u s e # 1 7
E L Q 2 7 1
C trl (F )
M o u s e # 1
M o u s e # 2
M o u s e # 3
T r e a t m e n t
1 5 8 1 2 1 5 1 9 2 3
C o n tro l 3
0 .0 0
0 .0 5
0 .1 0
2 .5
5 .0
2 5
5 0
7 5
E L Q 3 0 0 (1 0 m g /K g X 7 )
m o n ito r e d b y S m e a r
D a y s p o s t in fe c t io n
% P
ara
sit
em
ia
M o u s e # 1 6
M o u s e # 1 7
E L Q 3 0 0
C trl (F )
T r e a t m e n t
M o u s e # 4
M o u s e # 5
M o u s e # 6
1 5 8 1 2 1 5 1 9
C o n tro l 3
0 .0 0
0 .0 5
0 .1 0
2 .5
5 .0
2 5
5 0
7 5
E L Q 3 1 6 (1 0 m g /K g X 7 )
m o n ito r e d b y S m e a r
D a y s p o s t in fe c t io n
% P
ara
sit
em
ia
M o u s e # 1 6
M o u s e # 1 7
E L Q 3 1 6
C trl (F )
T r e a t m e n t
M o u s e # 7
M o u s e # 9
M o u s e # 8
1 5 8 1 2 1 5 1 9 2 3
C o n tro l 3
0 .0 0
0 .0 5
0 .1 0
2 .5
5 .0
2 5
5 0
7 5
E L Q 4 0 0 (1 0 m g /K g X 7 )
m o n ito r e d b y S m e a r
D a y s p o s t in fe c t io n
% P
ara
sit
em
ia
E L Q 4 0 0
C trl (M )
T re a tm e n t
M o u s e # 1 0
M o u s e # 1 1
M o u s e # 1 2
1
M o u s e # 1 7
M o u s e # 1 6
5 8 1 2 1 5 1 9
C o n tro l 3
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Figure 4
A C
B
LabS1-S
+
V
LabS1-S
+
ELQ-1
LabS1-S
+
ELQ-3
CL1
CL2
CL3
CL1
CL2
CL3
CL1
CL2
EL
Q-1
EL
Q-3
V
V2
71
-R
Pa
ra
sit
em
ia (
%)
26
DP
I
0 .0
0 .5
2 0
3 0
4 0
5 0
6 0
1
10
Pa
ra
sit
em
ia (
%)
39
DP
I
0 .0 0
0 .0 5
0 .1 0
2 0
3 0
4 0
5 0
6 0
10
LabS1-S
+
V
LabS1-S
+
ELQ-1
LabS1-S
+
ELQ-3
D
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19
Figure 5
Vannier, E. et al. (2012, New England Journal of Medicine)
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What is currently unknown?
• Effective treatment: Are Endochin-like
Quinolones the solution?
• Pathophysiology: why does recrudescence
occur?
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How to address the problem
of current therapies
• Prove that current therapies are ineffective and provide
evidence that new drug is significantly better than current
therapies
• Find out why current therapies do not work
• ELQs were considered potent due to previous studies
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Methodology of infecting mice
• In vivo study
• SCID Mice/Rag2 knockout mice: Mice with weaker
immune system
• Mice from Dr. Ruslan Medzhitov’s Lab
• 107 parasites
• 10mg/ kg by oral gavage 4 to 11 days post infection (dpi)
• Control mice given vehicle with no drug
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Methodology to test efficacy
• Blood collected from tail every 4 days
• Giesma Staining and Counting
• Flow cytometry to check counts
• DNA sequencing to identify mutations