The Difil Test kit for detection of canine heartworm microfilariae


Tilley, L.P.; Wilkins, R.J.

Veterinary Medicine, Small Animal Clinician 69(3): 288-294

1974


The Difil Test Kit appears to be a reliable working application of the filtration technique for the detection and differentiation of circulating microfilariae in the blood. The technical improvements and modification in lysing solution, staining technique and use of a clear transparent filter membrane have minimized residual cellular debris and eliminated many of the problems inherent in the original method. Because the filters effectively trap all microfilariae in the blood sample, quantitation of microfilariae recovered is possible, providing a known blood volume is used. In addition to its reliability, the filter technique is very rapid; no centrifugation is involved and little time is required to scan the slide. This method should allow practitioners to screen blood samples for circulating microfilariae quickly and accurately.

CLINICAL
0
1.
the
OIFIL
TEST
KIT*
for
detection
of
canine
heartworm
microfilariae
Larry
P.
Tilley,
D.V.M.
Robert
J.
Wilkins,
B.V.Sc.,
Dip.
V.P.
Animal
Medical
Center
510
East
62nd
Street
New
York,
New
York
10021
'Patent
pending
288
/
March
1974
0
F
THE
many
methods
described
for
de-
tecting
microfilariae
in
the
blood,
the
di-
rect
smear,
the
Knott's
test'
and
the
filter
technique
appear
to
be
most
widely
recom-
mended
2--,
and
used.
The
filter
technique,
as
originally
de-
scribed,"
uses
a
lysing
solution
containing
methylene
blue
(1:10,000).
After
this
solu-
tion
has
been
mixed
with
blood,
the
hemo-
lysed
specimen
is
injected
through
a
25
-mm.,
8
-micron
Millipore
fi
lter
pad
which
traps
the
microfilariae.
Several
technical
difficulties
have
been
encountered
with
this
filter
technique.
7
.
4
The
most
common
problem
is
that
some
samples
(approximately
20%)
fail
to
pass
through
the
fi
lters,
either
in
part
or
com-
pletely.
7
This
problem
has
been
attributed
to
fragmented
cell
ghosts,
nuclear
debris
and
mucoid
ground
substance
of
cytoplasmic
origin
which
readily
clog
the
pores
of
the
opaque
membranes.
Other
problems,
in-
cluding
overstaining
of
the
fi
lter
membrane,
poor
staining
of
microfilariae,
excessively
stained
cellular
debris,
rapid
drying
of
the
filter
membrane
and
excess
depth
of
field,
make
identification
and
differentiation
of
microfilariae
difficult.
A
commercially
available
test
kit,
the
Difil
Test
(EVSCO
Pharmaceutical
Corp.),
utilizing
the
principle
of
the
fi
ltration
tech-
nique,
overcomes
many
of
these
problems.
In
the
Difil
Test
method,
1
ml.
of
blood
is
drawn
directly
from
the
dog
and
immed-
iately
diluted
and
lysed
with
the
Difil
Test
lysing
solution,
which
contains
an
antico-
agulant.
Alternately,
if
performance
of
the
test
is
to
be
deferred,
blood
can
be
drawn
into
any
anticoagulant
such
as
EDTA,
he-
parin
or
sodium
citrate.
The
syringe
is
firmly
attached
to
the
ly-
sing-solution
dispenser
bottle
(Figure
1)
and
approximately
8
ml.
to
10
ml.
of
the
lysing
solution
is
aspirated.
The
amount
of
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and
analgesia.
Se-
dation
can
be
maintained
for
1
to
2
An
intramuscular
or
subcutaneou
ll
dosage
(1
mg.
per
pound
of
bod
weight)
produces
the
maximum
e
fect
within
10
to
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minutes;
intr
venous
dosage
(0.5
mg.)
within
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t
5
minutes.
Fast,
predictable
recovery.
Patier
emerges
as
if
waking
from
a
deep
sleet
usually
within
1
to
2
hours
after
injec
tion.
Post
-anesthetic
or
emergenc
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animals
preanesthetized
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This
material
was
copied
at
the
NUN
and
may
be
Subject
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Copyright
Laws
Cheri
gro
,r1
Figure
1
Approximately
1
ml.
of
blood
is
drawn
into
a
10-m1.
syringe
and
8
ml.
to
9
ml.
of
lysing
solution
is
aspirated.
Figure
2
The
hemolysed
specimen
is
agitated
to
lyse
both
red
and
white
cells.
Figure
3
The
clear,
colorless
filter
membrane
is
loaded
onto
the
gauze
mesh
of
the
filter
holder
and
the
cap
is
secured.
290
/
March
1974
voro-opr,
S
t
F
igure
4
(above)
—The
lysed
cell
solution
Is
Infected
slowly
through
the
filter
holder.
Figure
5
(above
right)
—One
or
two
flushes
With
Water
are
used
to
remove
excess
cellular
debris
from
the
filter.
Din!.TEST
KIT
(coNT'D)
f
ir
drawn
into
the
syringe
allows
for
mixing.
a
p
e
s
Yringe
is
agitated
four
or
five
times
to
cite
for
complete
lysis
of
both
red
and
does
cells
(Fi
gure
2).
The
lysing
solution
°1()
es
not
destroy
the
microfilariae.
b
A
s
pecially
designed
clear
fi
lter
mem-
ral
ne
is
lo
a
d
e
d
onto
the
gauze
mesh
of
t
h
e
fi
lter
h
o
ld
er
(Figure
3)
and
the
cap
is
firmly
secured.
With
the
syringe
c
o
nnected
to
the
f
ilter
ci
h
o
ld
er,
the
lysed
cell
solution
is
inject-
e
d
th
rough
the
system
(Figure
4).
fi1118
Procedure
is
followed
by
one
or
two
lishi
ngs
with
8
m
l
.
t
o
10
m
l
.
o
f
wa
t
e
r
to
el
ear
the
membrane
of
excess
debris
(Fig-
fil
To
avoid
flushing
trapped
micro
-
arise
through
the
membrane,
this
step
Figure
6
The
undersurface
of
the
filter
holder
is
blotted
to
drain
water
from
around
the
membrane.
The
cap
is
unscrewed
and
the
filter
is
removed,
as
in
Figure
3,
and
transferred
to
a
glass
slide.
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MEDICINE
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ANIMAL
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This
material
was
copied
atthe
NLM
and
maybe
March
1974
/
291
tE
Figure
7
Two
drops
of
the
stain
are
placed
on
the
filter
and
a
coverslip
is
applied.
Figure
8
Excess
stain
is
removed
by
pressing
the
coverslip
to
the
slide
between
a
gauze
pad.
4
DIFIL
TEST
KIT
(CONT'D)
must
be
performed
slowly.
The
undersurface
of
the
fi
lter
holder
is
blotted
to
drain
excess
water
from
around
the
filter
(Figure
6).
The
cap
is
removed
and
the
filter
gently
transferred
with
thumb
for-
ceps
to
a
clean
glass
slide,
keeping
the
fil-
tered
material
uppermost.
Staining
is
car-
ried
out
using
1
or
2
drops
of
Difil
Test
stain
(Figure
7).
A
coverslip
is
placed
over
the
stained
filter
and
excess
stain
is
re-
moved
by
pressing
the
coverslip
to
the
slide
between
a
gauze
pad
or
paper
towel.
The
preparation
is
then
ready
for
microscopic
examination
(Figure
8).
The
slide
can
be
rapidly
scanned
under
low
-power
magnification
(10X).
When
the
Difil
Test
technique
is
used,
microfilariae
stain
reddish
purple
(Figure
9).
They
may
be
differentiated
by
morphology
and
meas-
urement
of
length.
The
filter
holes,
which
are
visible
microscopically,
are
8
microns
in
diameter.
This
measurement
can
be
used
as
a
guide
in
determining
the
width
of
mi-
crofilariae
at
the
nerve
ring.
292
/
March
1974
VETERINARY
MEDICINE/SMALL
ANIMAL
CLINICIAN
The
length
of
microfilariae
of
Dirofilaria
immitis
and
Dipetylonema
reconditum
ap-
pears
shorter
with
this
method
than
pre-
viously
described."
Table
1
lists
the
differ-
ential
features
used
to
distinguish
the
two
species
of
canine
fi
lariae
when
this
method
is
used.
Length,
width
and
such
anatomic
features
as
the
tapered
or
blunt
head,
pres-
ence
or
absence
of
a
cephalic
hook:
or
button
-hook
tail,
are
the
major
features
by
which
microfilariae
may
be
differentiated.
Comparison
of
the
Filter
Technique
to
Other
Methods
Several
authors
have
published
data
com-
paring the other
common
methods
of
micro-
fi
lariae
detection
(Table
2)
to
the
filter
technique.
In
most
cases,
the
fi
ltration
technique
appears
to
be
more
accurate
in
detecting
microfilariae,
especially
when
the
numbers
of
parasites
in
the
circulating
blood
are
few.
12
This
is
true
except
in
reported
instances
7
.
8
when
the
technical
difficulties
previously
mentioned
led
the
authors
to
abandon
this
method.
Using
blood
samples
from
846
dogs
from
the
New
York
City
metropolitan
area,
our
laboratory
compared
the
direct
wet
smear
technique
to
the
modified
fi
ltration
test.
Of
the
846
samples,
27
(3.2%)
were
positive
by
TABLE
2
—Comparison
of
Filter
Techni
the
direct
smear,
while
34
(4.0%)
were
posi-
tive
using
the
Difil
Test.
The
Difil
Test
de-
tected
21%
more
dogs
with
heartworm
dis-
ease
than
did
the
conventional
method.
The
absence
of
circulating
microfilariae
in
the
blood
does
not
necessarily
exclude
the
possibility
of
heartworm
infestation.
In
approximately
5%
to
10%
of
the
dogs
with
D.
immitis
infestation,
microfilariae
cannot
be
detected
in
the
circulating
blood.''
Inade-
quate
sample
size,
immaturity,
sterility,
monogomy,
host
immunity
or
prior
therapy
are
some
of
the
reasons
for
these
false
neg-
ative
results.
When
heartworm
infestation
is
suspect
-
TABLE
1
—Canine
Microfilariae
Identification
Using
the
Filter
Technique
D.
reconditum
D.
immitis
Average
length
(microns)
213-240
240-273
Width
(microns)
4.7-5.8
6.1-7.2
at
nerve
ring
Narrower
than
As
wide
as
filter
holes
the
filter
holes
Anterior
extremity
Tail
Number
Cephalic
hook
parallel
button
-hooked
few
present
tapered
straight
many
absent
ue
to
Other
Methods
for
Detecting
Canine
Microfilariae
Author
Geographic
Region
Altman,
N.
H.
(1972)
Wylie,
J.
P.
(1970)
Wylie,
J.
P.
(1970)
Palumbo,
N.
E.
(1972)
Watson,
A.
D.
J.
(1973)
Wilkins,
R.
J.
(1973)
Ref.
No.
of
Dogs
Tested
Baltimore
2
1800
Boston
6
Boston
6
Hawaii
7
Sydney,
11
Australia
New
York
25
202
85
495
846
%
Positive
for
MIcrofilariae
(Filtration
Method)
°A,
Samples
Positive
for
the
Technique
but
Negative
Filtration
for:
Direct
Smear
Capillary
Technique
Knott
Method
26.7%
ND
5.0%
'
1.1%
100%
17%
18%
4%
25%
ND
ND
1.9°/0
23.5%
1.2%
ND
0%
8.1%
ND
ND
17.5%
4.0%
21%
ND
ND
ND
Not
Done
'Present
study
VETERINARY
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1974
/
293
0
elf
C
SE
416
Figure
9
—Stained
microfilaria
of
Dirofilaria
immitis
(x200).
Note
the
tapered
head
and
straight
tail.
Each
hole
in
the
filter
membrane
is
8
microns
in
diameter.
DIFIL
TEST
KIT
(CONT'D)
ed
clinically
we
have
found
collection
of
blood
samples
in
the
evening
to
be
benefi-
cial.
Other
diagnostic
tests
used
to
confirm
a
diagnosis
include
thoracic
radiographs,
electrocardiogram,
routine
blood
counts,
total
eosinophil
counts
and
serum
protein
electrophoresis.
Conclusion
The
Difil
Test
kit
appears
to
be
a
reliable
working
application
of
the
filtration
tech-
nique
for
the
detection
and
differentiation
of
circulating
microfilariae
in
the
blood.
The
technical
improvements
and
modifi-
cation
in
lysing
solution,
staining
technique
and
use
of
a
clear
transparent
fi
lter
mem-
brane
have
minimized
residual
cellular
de-
bris
and
eliminated
many
of
the
problems
inherent
in
the
original
method.
Because
the
filters
effectively
trap
all
microfilariae
in
the
blood
sample,
quantita-
tion
of
microfilariae
recovered
is
possible,
providing
a
known
blood
volume
is
used.
In
addition
to
its
reliability,
the
filter
technique
is
very
rapid;
no
centrifugation
is
involved
and
little
time
is
required
to
scan
the
slide.
This
method
should
allow
practi-
tioners
to
screen
blood
samples
for
circulat-
ing
microfilariae
quickly
and
accurately.
5
ACKNOWLEDGMENTS
The
technical
assistance
of
Miss
Denise
Vito
and
Mr.
Normal
Greenbury
is
acknowledged.
REFERENCES
1.
Newton,
W.L.;
Wright,
W.H.:
A
Re-evaluation
of
the
Canine
7";
1957.
Problem
in
the
United
States.
Vet.
Med.
52:75-
2.
Altman,
N.H.:
Canine
Ileartwortn
Disease;
Richard
Bradley.
ed.
University
of
Florida
Press,
1972;
pp.
87-93.
3.
Council
Report:
Procedures
for
the
Treatment
and
Preven.
tion
of
Canine
Ileartworm
Disease.
JAVMA
162:660-661;
I
Ic
-
,:;;
4.
Jackson,
R.F.:
Diagnosis
of
Heartworm
Disease
by
Examina
Lion
of
the
Blood.
JAVMA
/54:374-376;
1969.
5.
Kelly,
J.I).:
Detection
and
Differentiation
of
Microfilariae
in
Canine
Blood.
Austr.
Vet.
J.
49:23-27;
1973.
6.
Wylie,
J.I'.:
Detection
of
Microfilariae
by
a
Filter
Technique.
JAVMA
156:1403-1405;
1970.
7.
Palumbo,
N.E.;
Perri,
U.S.;
Some
Observations
on
Diagnosis
of
('((nine
Filariasis.
JAVMA
160:715-719;
1972.
ri
.
ri
tein.
F.J.;
Lawton,
G.W.:
Comparison
of
Methods
for
hag-
1 (
11;
;out
si
;
,,
1
7
7ferentiation
of
Canine
Filariasis.
-IA
V:11.-1
163
I
1,
1,
,
0.
Geurgi,
J.R.:
Parasitology
for
Veterinarians.
W.
B.
Saunders
(
Philadelphia,
l'a.,
1969;
pp.
42-43.
10.
1.i
ndsey,
.1.R.:
Identification
of
Canine
Microfilariae.
JA
VAX
-I
1.16:1106-111
,
1;
1965.
11.
Watson,
A.D.J.;
et
al.:
A
Comparison
of
Microfilariae
Iso-
hcted
from
Canine
Blood
by
the
Modified
Knott
Test
and
a
Filter
Method.
Austr.
Vet.
J.
49:28-30;
1973.
12.
Place,
M.A.:
Millipore
Technique
for
Identifying
D.
(Letters
to
Editor)
JAVMA
163:506;
1973.
13.
Wong,
M.M.;
et
al.;
Dirofilariasis
Without
Circulation
Micro-
fi
lariae—A
Problem
in
Diagnosis.
JAVMA
163:133-139;
1973.
294
/
March
1974
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