Diagnostic testing

Diagnostic testing
Relevant to:
1. Control and eradication campaigns
(e.g., serological screening;
genetic screening;
herd and flock accreditation)
2. Individual-animal clinical diagnosis
Diagnosis of infectious disease
in populations
1. Current infection
Clinical signs
Isolation of agent

Molecular changes
(surface antigens, integration)

IMMUNE RESPONSE
Biochemical changes
Diagnosis of infectious disease
in populations
2. Past infection
Clinical history
Pathognomonic changes
IMMUNE RESPONSE
SEROLOGICAL EPIDEMIOLOGY
Strictly: any variable in serum
(e.g., metabolites)

Conventionally: antibodies
Nature of veterinary data
Problem of variability
E.g. Milk yield
Weight
Height
Antibody titres
Data
Quantitative
Nominal Ordinal Discrete
(counts)
Continuous
(measurements)
Interval Ratio
Qualitative
(categorical)
Variability:
Demonstrated in single samples
for continuous data (and ordinal
data)

16

14

12

10

8

6

4

2

0
1 2 3 4 5 6 7
Weight (kg)
N
u
m
b
e
r

o
f

p
i
g
s

Median (Q
2
) = 50
th
percentile
Standard deviation (s)
Central tendancy
Spread
= (x)
n
Mean (x)
Where s
2
= (x- )
2

(n-1)
x
Semi-interquartile range (SIR)
SIR = (Q
3
-Q
1
)
2
Where
Q
1
= 25
th
percentile (lower quartile)
Q
3
= 75
th
percentile (upper quartile)
-3 -2 -1 0 1 2 3
P
r
o
p
o
r
t
i
o
n

o
f

v
a
l
u
e
s

o
f

x
/
u
n
i
t

0.1
0.2
0.3
0.4
0.5
0.6
0.7
x
Normal distribution
t distribution
lognormal distribution
Antibody dilution:
Usually with a constant ratio
between dilutions (i.e. geometric)
This allows measurement on a
logarithmic scale, because:
1. Antibodies are lognormal
2. Logs
2
can be used as coded values









N
u
m
b
e
r

o
f

b
i
r
d
s

Antibody titres (logarithmic scale)
0
<10
35 -
40 -
25 -
20 -
15 -
5-
30 -
10 -
10 20 40 80 160 320 640 1280 2560 >2560
Antibody titres expressed as reciprocal
dilutions (X) and coded titres (log
2
X)
Reciprocal dilution
(X)
Coded titre
(log
2
X)
1 (undiluted serum) 0
2 1
4 2
8 3
16 4
32 5
64 6
Mean titres
Arithmetic mean
Mean of coded titres:
E.g. five titres 1/2, 1/4, 1/2, 1/8, 1/4
Coded as: 1, 2, 1, 3, 2
Arithmetic mean = (1+2+1+3+2)/5
= 1.8
Geometric mean
Antilog
2
of arithmetic mean
i.e. 2
1.8
= 3.48
NB Sernonegative animals CANNOT be included
Why cant seronegative
animals be included?
Because the logarithm of zero is:
-
INTERPRETATION OF TESTS
Refinement
(Analytical specificity)
Accuracy
The classification of some influenza A viruses
(based on Murphy and Webster, 1990)
Haemagglutinins (H) and
neuraminidases (N)
Strains
H1 N1 PR/8/34
H1 N1 Sw/Ia/15/30
H2 N2 Sing/1/57
H3 N2 HK/1/68
H3 N2 Sw/Taiwan/70
H3 N8 Eq/Miami/1/63
H4 N6 Dk/Cz/56
H5 N3 Tern/S.A./61
H6 N2 Ty/Mass/3740/65
H7 N7 Eq/Prague/1/56
Accuracy
(Results in the individual animal)
Positive results:
Actual infection True +ve
Cross-reactions False +ve
Negative results:
Absence of infection True -ve
Improper test False -ve
Improper timing ..
Immunotolerance ..
Inhibitors ..
Insensitive tests* ..
* Analytical sensitivity
EVALUATION OF DIAGNOSTIC TESTS
Validity
Reliability (repeatability)
Both are long-run properties
1 2 3 4 5 6 7 8 9 10
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9 10
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9 10
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9 10
1
2
3
4
5
6
7
8
9
1 2 3 4 5 6 7 8 9
1
2
3
4
5
6
7
8
9
Validity
High
H
i
g
h

Low
L
o
w

A B
C D
Test status True
+ve
Status -ve Total
+ve a b a + b
- ve c d c + d
Total a + c b + d
Sensitivity* = a/a + c
Specificity* = d/b + d
Fully:
Diagnostic sensitivity
Diagnostic specificity
Organ Condition Sensitivity
(%)
Liver Fascioliasis 19.07
Lung Tuberculosis 5.55
Tongue and
muscle
Cysticercosis 0.00
Sensitivity of beef inspection
procedures (Nigerian abattoir)
Test status True status Totals
Positive Negative
Flotation positive 22(a) 1(b) 23
Flotation negative 14(c) 43(d) 57
Totals 36 44
Possible results of a diagnostic test exemplified by
application of a centrifugation/flotation technique to
horses of known tapeworm status
(from Proudman and Edwards, 1992)
Sensitivity = a/(a + c) = 22/36 = 0.61 (61%)
Specificity = d/(b + d) = 43/44 = 0.98 (98%)
Test Sensitivity Specificity
2G6 Ag-ELISA 71% 75%
Tr7 Ag-ELISA 81% 78%
IgG ELISA 89% 92%
Trypanosoma evansi
Diagnostic tests
Microhaematocrit/mouse inoculation/
card agglutination test/ELISAs
NB. Two types of sensitivity
and specificity
1. Diagnostic sensitivity and
Diagnostic specificity
Potential for confusion!
2. Analytical sensitivity and
Analytical specificity
Prevalence correction for
misclassification
Test prevalence = P
T
True prevalence = P

P =

P
T
+ specificity - 1

Sensitivity + specificity - 1

Test status
1. Discrete category
(e.g. +ve or ve to radiography;
presence or absence of a clinical sign)
May be based on:
2. Continuous variable
(e.g. blood enzyme level; antibody
titre)









N
u
m
b
e
r

o
f

b
i
r
d
s

Antibody titres (logarithmic scale)
0
<10
35 -
40 -
25 -
20 -
15 -
5-
30 -
10 -
10 20 40 80 160 320 640 1280 2560 >2560
Continuous variables:
Require definition of a
cut-off value
FMD: OIE protocol
Duplicate wells:
Blocking ELISA:
Positive if mean 1:45
E.g. Well 1:1/32
Coded titre = 5
Well 2:1/64
Coded titre = 6
AMT = (5+6)/2
= 5.5
GMT = 2
5.5
= 45.25
Mean titre = 1:45
Specificity
Sensitivity
C
Effect of various positive thresholds on the serodiagnostic
interpretation of the enzyme immunoassay for the detection
of bovine antibody to Brucella abortus
Positive
threshold
(OD 414 nm)
Sensitivity Specificity Prevalence
of disease
Predictive value

Non-vac

Vac
(S19)
+ve test -ve test
Either
Non-vac Vacc
0.220 0.960 0.990 0.852 0.1
0.01
0.001
0.92
0.50
0.09
0.42
0.06
0.01
1.00
1.00
1.00
0.260 0.943 0.995 0.930 0.1
0.01
0.001
0.95
0.64
0.15
0.60
0.12
0.01
0.99
1.00
1.00
0.300 0.937 0.998 0.948 0.1
0.01
0.001
0.98
0.84
0.34
0.67
0.15
0.02
0.99
1.00
1.00
0.340 0.920 0.999 0.969 0.1
0.01
0.001
0.99
0.90
0.48
0.77
0.23
0.03
0.99
1.00
1.00
Defining a cut-off point for continuous
and ordinal test variables
1. 2 or 3 standard deviations greater than the mean
in unaffected animals (does not consider
distribution in affected population)
Several approaches:
2. 5
th
percentile in affected animals (does not
consider distribution in healthy population)
3. Minimum number of incorrect diagnoses
4. Minimum cost of incorrect diagnoses
ROC curve
(100-Specificity) (%)
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70 80 90 100
S
e
n
s
i
t
i
v
i
t
y

(
%
)

A
Definition of true status
True positive
Requires a gold standard
1. Experimental infections
2. Several tests (+ve to all)
True negatives
1. Animals from countries where infection is known
to be absent
2. Several tests (-ve to all)
Sensitivity and specificity
Precision
Required (confidence intervals)
Rule of thumb:
100 True +ves
200 True -ves
Possible results of a diagnostic test
Test status True status Totals
Diseased Not diseased
Diseased a b a + b
Not diseased c d c + d
Totals a + c b + d a + b + c + d
Test status True status Totals
+ve -ve
+ve a b a + b
-ve c d c + d
Totals a + c b + d
Sensitivity = a/(a +c)
Specificity = d/(b + d)
Predictive values
+ve test result = a/(a + b)
-ve test result = d/(c + d)
Sensitivity and specificity of three
screening tests for bovine brucellosis
Sensitivity
(%)
Specificity
(%)
Tube agglutination test 62.0 99.5
Complement fixation test 97.5 99.0
Brewer card test 95.2 98.5
Predictive value (positive result) of the tube agglutination test
for bovine brucellosis at three different prevalence levels
Test status True status Totals
Brucellosis
present
Brucellosis
absent
Brucellosis
present
1860 (a) 485 (b) 2345
Brucellosis
absent
1140 (c) 96515 (d) 97655
Totals 3000 97000 100000
Predictive value (positive result) =
(Sensitivity = 62% Specificity = 99.5%)
(Prevalence of brucellosis: 3%
a
a + b
= 79.5%
Prevalence of brucellosis: 0.1%
Test status True status Totals
Brucellosis
present
Brucellosis
absent
Brucellosis
present
60 500 560
Brucellosis
absent
40 99400 99440
Totals 100 99900 100000
Predictive value (positive result) = 10.7%
Prevalence of brucellosis: 0.01%
Test status True status Totals
Brucellosis
present
Brucellosis
absent
Brucellosis
present
6 500 506
Brucellosis
absent
4 99490 99494
Totals 10 99990 100000
Predictive value (positive result) = 1.2%
Aggregate (herd-level) testing
(1) Aggregate sensitivity
Se
agg
= 1-(1-P
T
)
n
P
T
= test prevalence
E.g.: True prevalence, P, = 0.25
n

= number of animals sampled
Se = 0.97,
Sp = 0.98,
n = 4
P
T
= {P x sensitivity} + {(1 P) x (1 specificity)}
= {0.25 x 0.97} + {(1 0.25) x (1 0.98)}
= 0.2575
Se
agg
= 1 (1 012575)
4
= 1 0.7425
4
= 1 0.3039
= 0.70
I.e. 70% of all affected herds have one or more test-positive
animals (30% of affected herds are missed)
Aggregate (herd-level) testing
(2) Aggregate specificity
Sp
agg
= (Specificity)
n
E.g.:
Sp = 0.98
n = 4
Sp
agg
= 0.98
4
= 0.92

I.e. 92% of all unaffected herds have no test-positive
animals in each sample of 4 animals.
(8% of unaffected herds will test positive)
Tests at aggregate (herd) level
Net effects
As the number of animals tested increases

Aggregate (herd) specificity
Aggregate (herd) sensitivity
Multiple testing strategies
Two main types
Parallel
Serial
Multiple testing strategies
Parallel
Positive to any test
e.g. Brucellosis
Tests:
1. Bacterial culture
2. Rose Bengal test
3. Milk ring test
Multiple testing strategies
Serial
Positive to all tests
e.g. Swine dysentery
Tests:
First fluorescent antibody test on faecal
or mucosal smears
Then positive confirmed by bacterial
culture
Multiple testing strategies:
sample calculations
Example
Test A: Se 80%; Sp 60%
Test B: Se 90; Sp 90%
True prevalence = 0.20 (20%)
True proportion negative = 80%
Multiple testing strategies:
sample calculations: PARALLEL
Sensitivity
Test A detects 80%, leaving 20% undetected
Test B detects 90% of the remaining 20% = 18%
Therefore total +ve to either test = 80% + 18%
= 98% = parallel Sensitivity
Specificity
Test A categorises 60% of true negatives as negative
Test B categorises only 90% of these as negative
i.e. 90 x 60% = 54%, negative to both
= parallel Specificity
Multiple testing strategies:
sample calculations
Parallel testing
Complete the contingency table:
Positive predictive value: 19.6/56.4 = 35%
True +
(%)
True -
(%)
Total
Test +(either) (%) 19.6 36.8 56.4
Test - (both) (%) 0.4 43.2 43.6
Total 20 80 100
Negative predictive value: 43.2/43.6 = 99%
Multiple testing strategies:
sample calculations:SERIAL
Sensitivity
Test A detects 80%
Test B detects 90% of these as positive
Therefore % positive to both tests
= 80% x 90% = 72% = Serial Sensitivity
Specificity
Test A correctly categorises 60% of true negatives,
leaving 40%
Test B categorises 90% of these as negative
= 90% x 40% = 36%, negative
= Serial Specificity
Therefore total number negative to either test
= 60% + 36% = 96%
Multiple testing strategies:
sample calculations
Serial testing
Complete the contingency table:
Positive predictive value: 14.4/17.6 = 82%
True +
(%)
True -
(%)
Total
Test +(both) (%) 14.4 3.2 17.6
Test - (either) (%) 5.6 76.8 82.4
Total 20 80 100
Negative predictive value: 76.8/82.4 = 93%
Multiple testing strategies:

Third type
Repeated testing (negative-herd retesting)
Exclusive to veterinary medicine
Applied to herds not individuals
Using the same test e.g. tuberculosis
E.g., Comparative intradermal tuberculin test:
1. Reactors culled;
2. -ve and inconclusive reactors tested every 60
days until two clear herd tests are achieved;
3. Then return to 4-year testing cycle