1. 2 cryptosporidium volunteer study investigators: cynthia chappell, phd pablo okhuysen, md herbert...
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Cryptosporidium parvum: Infectivity in Healthy Volunteers and Laboratory Animal Models
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Cryptosporidium Volunteer Study
Investigators:Cynthia Chappell, PhDPablo Okhuysen, MDHerbert DuPont, MD
Isolates:Charles Sterling, PhD (Iowa)Karen Snowden, DVM (TAMU)Joseph Crabb, PhD (UCP)
Laboratory staff:Blue JohnsonHan DangConstance WangMichael ColettaSonia BakerDanny NguyenMarilyn Marshall
UCRC Nursing staff:Madeline Jewell, RNJulie Rice, RNNai-Hui Chiu , RN
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Inclusion criteria
Cryptosporidium serum IgM/IgG statusNegative history and physical
ElectrocardiogramChest X-rayStool for occult blood and parasitesBlood chemistry (SMAC)Complete blood countUrinalysis
Pregnancy testHepatitis BsAgHepatitis CImmunoglobulin levelsSyphilis (RPR)HIVT-cell subsets
Pass written examination documenting understanding of study.
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Infant under 2 years in householdElderly person in householdAny contact with immunocompromised persons
Exclusion criteria
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Gender (%):
Male 53.1 Female 46.9
Ethnicity (%):
Caucasian 59.2Hispanic 15.4Black 13.8 Asian 10.0Other 1.5
Volunteer Study Population (n=130)
18-2
5
26-3
0
31-3
5
36-4
0
41-4
5
45-5
0
>50
Years of age
0
10
20
30
40
Per
cen
t
6
Preparation of Challenge Dose
Passaged in calf and purified
Shipped to Houston in potassium dichromate
Tested for adventitious agents (viruses, bacteria)
Adjusted for appropriate number
Delivered via gelatin capsule
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Specifications for oocyst use
Negative microbiology (no viable adventitious agents)
80+ excystation rate
Within 6 weeks of passage
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Study design
Oocyst collection
Calf infection
Oocyst purification
CD1 neonatal mouse
HCT-8 cell cultures
Healthy volunteers
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Isolate HistoriesIOWA isolate:
Collected from calf with diarrhea (Dr. Harley Moon, University of Iowa)Virulent for calves and humans after multiple passages Stable genotype 2 (multilocus analysis)
TAMU isolate:Identified in a foal with severe diarrhea Oocysts collected from stool student who contracted infection at necropsy Amplified in calfStable genotype 2 (multilocus analysis)
UCP isolate: Collected from calf with diarrhea (Maine)Virulent for calves after multiple passages Stable genotype 2 (multilocus analysis)
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Volunteer Study Design
Good general healthNormal immune status
normal IgA levelsnormal T-cell subsets
HIV-negativeC. parvum ab status (ELISA)
Challenged with a single dose of C. parvum oocysts
Volunteers selected for:
Stool collection(all for 14d, 2/wk for 6 wks)
Physical exam(daily for 14d, 2/wk for 6 wks)Personal health diary, including all symptoms
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0 1 2 3 4 5
Challenge dose (log)
20
30
40
50
60
70
80
90
100
110C
um
ula
tive
% in
fect
ed
IOWATAMUUCP
Ref of method: Reed and Muench, Am J Hyg 27:493, 1938
ID50 in Antibody-negative Volunteers
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Diarrhea
51.7
Oocysts
20.7
Neither
27.6
IOWA (n=29)Diarrhea
58.8
Oocysts5.9
Neither
35.3
UCP (n=17)
Diarrhea85.7
Neither14.3
TAMU (n=14)
Clinical Outcome in Volunteers
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0
10
20
30
40
50
60
70
80
90
ILL
NE
SS
AT
TAC
K R
AT
E (
%)
UCP
IOWA
TAMU
Illness Parameters Among C. parvum Isolates
Incubation period (5-9 days)Duration of diarrhea (2.7-4.4 days)Total # unformed stools (6.7-10.6)Total unformed stool weight (798-1278 g; highest with TAMU)
Not significant: p=0.045
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CD1 neonatal mouse model
CD1 mice (4-6 days old) orally gavaged with single dose of C. parvum oocysts
Neonates raised by their dams until termination of experiment at 7 days PI
Infection assessed by microscopy of terminal ileum
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0 0.5 1 1.5 2 2.5 3
Challenge dose (log)
0
20
40
60
80
100
120
Cu
mu
lati
ve
% i
nfe
cti
on
IowaTamuUCP
Dose Response Curves in Neonatal Mice
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HCT-8 (human enterocyte) cell cultures
HCT-8 cells placed in well (1000 cells/mm2)
Grown to 70-80% confluency (approx. 18 hrs)
Oocysts permeabilized with sodium hypochlorite
Oocysts added to cells in 1:1 ratio
Infection developed for 24 hrs
Fixed with cold MEOH
Stained and counted
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Cryptosporidium Infection in HCT-8 Cells
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C. parvum infectivity in HCT-8 Cells
0
10
20
30
40
50
60
70
% I
nfe
cti
vit
y
Isolates tested in 4 experiments (each in triplicate)
p=<0.01
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0.5 1 1.5 2 2.5 3 3.5
Volunteer ID50 (log)
20
30
40
50
60
70
Per
cen
t in
fect
ion
0.5 1 1.5 2 2.5 3 3.5
Volunteer ID50 (log)
1.4
1.5
1.6
1.7
1.8
1.9
2
2.1
Mo
use
ID50
(lo
g)
Infectivity in Human vs Neonatal Mice and HCT-8 Cells
r2 = 0.933 r2 = 0.548
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Conclusions
C. parvum oocysts vary widely in infectivity, even within the same genotype
HCT-8, a human enterocyte cell line, shows a higher correlation with human genotype 2 infectivity than does the neonatal mouse model.
HCT-8 also has the advantage of supporting genotype 1 replication.