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Journal of Clinical Microbiology, September 1999, p. 2931-2935, Vol. 37, No. 9
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Detection of Leishmania infantum in Dogs
by PCR with Lymph Node Aspirates and Blood
S.
Reale,*
L.
Maxia,
F.
Vitale,
N. S.
Glorioso,
S.
Caracappa, and
G.
Vesco
Istituto Zooprofilattico Sperimentale Della
Sicilia, Palermo, Italy
Received 20 January 1999/Returned for modification 14 April
1999/Accepted 15 June 1999
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ABSTRACT |
The PCR technique was applied to the diagnosis of leishmaniasis in
dogs, both serologically negative and positive. DNA was taken from
lymph node aspirates and blood. The primers 13a and 13b, derived from
Leishmania amazonies and Leishmania
braziliensis kinetoplast DNA (kDNA), also amplified
Leishmania infantum IPT1 constant region of minicircle
kDNA. The amplified fragment is 116 bp long. It was cloned and the
sequence was determined. A 70-bp inner fragment was designed and used
as a probe in dot blot hybridization. A group of 124 dogs was examined,
37 of which showed typical symptoms of disease. PCR was performed on
124 blood samples and 52 lymph node aspirates. Using microscopic
examination as the "gold standard," we calculated sensitivity,
specificity, and positive and negative predictive values of 100% using
lymph node aspirates and values of 85, 80, 95, and 57%, respectively,
using blood samples. We found that 40% of the animals without lesions and 38% of the animals with clinical signs gave false-negative results
by indirect immunofluorescence antibody testing. These animals could
contribute to the spreading of infection among dogs, and represent a
potential risk for human health as well.
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INTRODUCTION |
Leishmaniasis is a typical example
of zoonosis found on all the continents except Australia and Antarctica
(3). The geographic distribution depends on the presence of
sandfly vectors and animal reservoirs. In Southern Europe, canines are
considered the main reservoir of infection and phlebotomies are the
vectors. In Sicily, as in all the Mediterranean areas, sandflies are
present during the great part of the year because its climatic
situation permits the perpetuation of the sandfly life cycle
(9-11). Visceral leishmaniasis is becoming a real problem
of public health because it is an opportunistic infection in
immunocompromised patients and in human immunodeficiency virus-positive
subjects, especially in areas where leishmaniasis is endemic
(14-18). In Italy, the visceral form of disease is due exclusively to Leishmania infantum ZMON1, and its prevalence
is growing (15, 16). The parasites can be isolated from
either infected organs or lesions and can be cultivated in vitro.
Different tests, such as the indirect immunofluorescence antibody test
(IFAT), the enzyme-linked immunosorbent assay (ELISA), microscopic
examination of smears, and cultural isolation, are routinely used for
diagnosis. The immune reaction assays depend on the general health
conditions of the animal and false-negative results cannot be excluded,
so microbiological techniques, direct microscopic examination, or growth in axenic cultures remain the "gold standards" for
definitive diagnoses. Unfortunately, they take a long time and can fail
when the parasite load in the sample is light. However, significant progress in the development of new techniques for diagnosis and epidemiological studies has been made.
PCR has been applied as an analytical method to amplify a short
sequence in the minicircles and to reveal the presence of small numbers
of parasites directly in samples (8, 18, 19, 23-28,
34-36). In this study, we describe the use of PCR as a
diagnostic tool for canine leishmaniasis. Two different kinds of
samples (blood and lymph node aspirate) from the same animals were
compared. Moreover, correlation between serological, direct
examination, culture isolation, and PCR results was made. It is known
that the L. infantum genome comprises 36 chromosomes and a
variably sized DNA maxicircle (20 to 40 kb) and minicircle (1 to 2 kb) named kinetoplast DNA (kDNA) (7). The minicircle sequences show a variable region of about 600 bp and a constant region of about
200 bp conserved among the different Old and New World species (6). We used primers described by Rodgers et al.
(27) that amplify the conserved region in the minicircle
kDNA of different Leishmania species (27, 32). As
the homology of the constant region isn't total among all the species
and the complete sequence of L. infantum ZMON1 kDNA isn't
known, here we present the sequence of this amplified fragment.
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MATERIALS AND METHODS |
Direct examination of smears.
Slides were stained with
Giemsa and examined with a microscope using a 100× oil objective. Two
series of observation were performed and approximately 100 microscopic
fields were examined for each sample.
Parasite cultures.
The reference strain was IPT1 ZMON1 from
the collection of the Istituto Superiore di Sanità (Rome, Italy).
The IPT1 and the isolated strains were grown in Tobie agar medium
(33) modified by Evans with 15% rabbit blood, 5% fetal
bovine serum, 250 µg of gentamicin/ml, and 500 µg of
5-fluorocitosine/ml. The cultures were incubated at 25°C for 7 days.
In case of a negative culture result, 1 ml of the culture sample was
subcultured in the medium for another 10 days to confirm the absence of
the parasite.
IFAT.
The IFAT was performed in accordance with the methods
of Badaro et al. (5) and Duxbury and Sadun (13)
with some modifications. The IPT1 ZMON1 strain was used as antigen
fixed on multispot microscope slides (BioMerieux) in acetone bath. The
dog sera and positive and negative controls were diluted 1:80 in
phosphate-buffered saline (PBS) buffer. Aliquots of 10 µl were
spotted on each circle and the slides were incubated for 10 min at
37°C with 95% humidity. Fluorescent staining was performed by using
a fluorescein-labelled anti-dog gamma globulin (BioMakor) diluted 1:100
and colored with 0.002% Evans blue-PBS solution. The positive control
consisted of a known-titer serum from an IFAT-positive dog with
positive cultural isolation. The negative control consisted of
IFAT-negative dog serum from an animal that tested negative by ELISA
and culture test; both controls were purchased by Istituto Superiore di
Sanità. Another control consisted of the PBS-diluted (1:100)
anti-dog gamma globulin. Visualization was carried out with a Leica
DMLB microscope. The IFAT result was regarded as positive if a 1:80 dilution of the serum gave an evident yellow-green fluorescent signal
upon microscopic observation, while nonreactive samples appeared
red-brown.
DNA extraction.
DNA extraction from parasite cultures was
carried out as described by Schonian et al. (31), with some
modifications. The parasite culture was centrifuged for 15 min
(3,500 × g, 4°C) in a GPR Beckman centrifuge and
resuspended in lysis buffer (50 mM NaCl, 10 mM EDTA, 50 mM Tris-HCl
[pH 7.4]) with 1% sodium dodecyl sulfate (SDS). The mixture was
incubated for 30 min at 60°C, and proteinase K (Sigma Chemical Co.)
was then added to a final concentration of 0.1 mg/ml and the incubation
proceeded for 3 h at 60°C. After double extraction with buffered
phenol and with chloroform-isoamyl alcohol (25:24:1 [vol/vol/vol]),
the DNA was precipitated with sodium acetate (0.3 M final
concentration), overlaid with 2.5 volumes of ice-cold ethanol, and
mixed by inversion. DNA was recovered by centrifugation at
13,800 × g for 20 min, washed with 70% ethanol (vol/vol), and suspended in distilled water. The DNA was incubated with
RNase A (Boehringer Mannheim) (100 µg/ml) at 37°C for 1 h and
further purified by phenol-chloroform extraction and a precipitation step. The concentration and purity of extracted DNA were calculated by
reading A260 and A280
with a Hitachi U-1100 spectrophotometer. All reagents were supplied by
Sigma Chemical Co.
DNA purification from dog samples.
DNA was extracted from
samples of blood and lymph node aspirate. The extraction from blood was
performed with a Rapid Prep Genomic DNA Isolation Kit (Pharmacia
Biotech). The blood samples (0.5 ml) were briefly incubated with 1 volume of suitable ice-cold lysis buffer, centrifuged, and washed with
another volume of water diluted 1:1 (vol/vol) with ice-cold lysis
buffer. After incubation at 55°C for 10 min with 50 µl of
extraction buffer containing guanidinium isothiocyanate, the samples
were applied to the spin columns, which were then centrifuged. After
two rounds of washing, the DNA was eluted with the appropriate buffer,
precipitated in 0.8 volumes of isopropanol, centrifuged at
16,000 × g, and suspended in 50 µl of sterile
distilled water. The QIAamp Blood and Tissue Kit was employed for
extraction of DNA from lymph node aspirates. Lymph node aspirates were
diluted with 0.5 ml of PBS solution, and 200 µl of this mixture was
incubated in a suitable lysis buffer with 25 µl of proteinase K (20 mg/ml) for 5 min. After vortexing, the mixture was incubated at 70°C
for 10 min, and ethanol (0.52 volumes) was added. The mixtures were
applied to the QIAamp spin columns and centrifuged. After two rounds of
washing, DNA was eluted with 200 µl of the supplied buffer preheated
to 70°C.
DNA amplification by PCR.
The target DNA for amplification
is a 116-bp fragment in the constant region of the kDNA minicircle.
This is one of the kDNA minicircle families currently used to identify
the Leishmania genus.
The primers used were a pair of oligonucleotides described by Rodgers
et al. (27), 13a (5'-dGTGGGGGAGGGGCGTTCT-3') and
13b (5'-dATTTTACACCAACCCCCAGTT-3'). Within the 116-bp
fragment, we designated a specific probe of 70 bp, the sequence of
which is presented in Fig. 2. The oligonucleotides for PCR and the
probe were supplied by Cruachem-Celbio. PCR amplification was carried out in 100-µl reactions containing (final concentrations) 4.0 mM
MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 9.3), 0.01% Triton
X-100, the four deoxynucleoside triphosphates (250 µM each), 400 nM
each primer, 10 µl of template DNA solution, and 2.5 U of DNA
Taq polymerase (Finnzyme). The reactions were performed in
an automated thermal cycler (Mini Cycler; MJ Research, Inc.). The
conditions were set as follows: denaturation at 94°C for 1 min,
annealing at 60°C for 1 min, and extension at 70°C for 1 min. A
7-min extension period at 72°C was added after 30 cycles. A positive
control containing 10 ng of genomic Leishmania DNA, and a
negative control without template DNA were included.
Electrophoresis.
Purified DNA and PCR products were analyzed
by electrophoresis through 1% and 2.2% neutral agarose gels,
respectively, containing 0.1 µg of ethidium bromide (Bio-Rad
Laboratories)/ml in TBE buffer (0.089 M Tris-HCl-0.089 M boric
acid-0.002 M EDTA) (30). The gels were visualized under UV
light with a transilluminator (UV-GENTM; Bio-Rad Laboratories) and
photographed with Polaroid 667 film. The DNA markers used were
/HindIII and ladder 50 (Pharmacia) for high and low
molecular weights, respectively.
Cloning and sequencing of 116-bp fragment.
The 116-bp
fragment obtained by PCR was cloned using a TA cloning kit
(Invitrogen). The recombinant plasmid was obtained by ligation of 50 ng
of pCR 2.1 vector with 10 ng of fresh PCR product. This plasmid was
used to transform XL One Blue supercompetent cells. Briefly, 2 µl of
0.5 M 
-mercaptoethanol and 2 µl of the ligase reaction product
were added to 100-µl vials of frozen cells. After 30 min on ice, the
cells were heat shocked for 30 s in a 42°C water bath and
incubated in 900 µl of preheated SOC medium at 37°C for 1 h
with 225-rpm rotary shaking. Aliquots of 50, 100, 200, and 300 µl
from each transformation were spread on Luria-Bertani plate agar
containing 50 µg of ampicillin/ml, 30 µl of 20-mg/ml X-Gal
(5-bromo-4-chloro-3-indolyl--D-galactopyranoside) and
IPTG (isopropyl--D-thiogalactopyranoside) solutions. The
plates were incubated for 18 h at 37°C. The picked colonies were
grown overnight in 20 ml of Terrific Broth. The plasmids were extracted
as described in the protocol of Qiaquick extraction kit (Qiagen). The
recombinant plasmids were revealed by double digestion with
HindIII-EcoRV restriction enzyme in
accordance with the manufacturer's instructions (Boehringer). The
116-bp fragment was sequenced by PCR using the M13 forward primer
within the plasmidic vector as the reaction start point. The reactions
were performed by a sequencing service in M-Medical laboratory, La
Sapienza University (Rome, Italy).
DNA labelling of the 70-bp DNA probe.
A 70-bp inner fragment
was designed by sequencing the amplified fragment. The probe was
labelled with digoxigenin (DIG)-dUTP by using a DIG DNA Labelling Kit
(Boehringer Mannheim) according to the instructions of the
manufacturer. For each reaction, 60 ng of DNA was labelled at 37°C
overnight and employed in hybridization.
Dot blot assay.
The amplified DNA was denatured for 5 min at
100°C and kept on ice. Samples of 5 µl were spotted on positively
charged nylon membrane (Hybond-N; Amersham) and fixed by UV exposure at
260 nm for 3 min. The blots were treated with 0.4 M NaOH for 3 min and
neutralized with 1 M Tris-HCl (pH 8.0) for 3 min before starting the
prehybridization (30).
Hybridization and detection.
The filter was hybridized at
60°C overnight in an incubation bag. The prehybridization mix
consisted of 4× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium
citrate), 5× Denhardt solution (2% Ficoll, 2% albumin, bovine
fraction V, 2% polyvinylpyrrolidone), and 1% SDS. About 4 ml of
hybridization solution were used per 100-cm2 membrane. The
hybridization solution contained 30 ng of the freshly denatured (10 min, 100°C) DIG-dUTP-labelled 70-bp probe/ml. After incubation, the
membrane was washed twice in 1× SSC-1% SDS (100 ml/100-cm2 membrane) for 15 min and twice in 1× SSC-0.1%
SDS at 65°C for 15 min. The presence of a DIG-labelled probe was
detected by using the alkaline phosphatase-conjugated antibody and CSPD
substrate according to the instructions of DIG-dUTP DNA detection kit
(Boehringer Mannheim). The chemiluminescent signal was revealed on
X-ray film (X AR OMAT; Kodak) after exposure for 10 min at room temperature.
Nucleotide sequence accession number.
After comparison with
correlated sequence, the 116-bp fragment was submitted to GenBank and
was assigned accession no. AJ131633.
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RESULTS |
Specificity and sensitivity of PCR.
The primers 13a and 13b
were used for PCR analysis of purified DNAs from Trypanosoma
equiperdum, Trypanosoma cruzi, and L. infantum IPT1 ZMON1. Moreover, some blood and lymph node aspirate samples taken from healthy dogs living in Bolzano, Italy, where any
case of leishmaniasis was recorded, were included in the specificity test. A 116-bp amplified fragment and hybridization signal obtained by
Southern blotting was seen only in Leishmania species (data not shown).
The sensitivity of the PCR was determined by adding mixtures containing
decreasing amounts of
L. infantum genomic DNA in a
range
between 1 ng and 0.01 fg to the reaction vials. Fig.
1 shows
that 0.01 fg of total DNA was
amplified and in Southern blot analysis
gave a detectable hybridization
signal with the 70-bp probe. The
sequence of the 116-bp amplified
fragments is shown in Fig.
2.
It is the
L. infantum ZMON1 specific fragment inside the constant
region of kinetoplast minicircle spacing between 13a and 13b primers.
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FIG. 1.
Sensitivity of DNA detection by Southern blot analysis
after PCR of purified L. infantum DNA. Serial dilutions from
1 ng to 0.01 fg of purified L. infantum DNA were amplified
by PCR, and products were analyzed by 2.5% agarose gel
electrophoresis. Hybridization of the 116-bp amplified fragment was
performed with a 70-bp labelled probe. Details of PCR amplification,
sequence of primers, cloning, and sequencing of probe are given in the
text. Lanes 1 to 9: 1 ng, 0.1 ng, 0.01 ng, 1 pg, 0.1 pg, 0.01 pg, 1 fg,
0.1 fg, and 0.01 fg, respectively, of DNA; lane 10, control without
L. infantum DNA.
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FIG. 2.
L. infantum sequence of 116-bp amplified
fragment within the kDNA constant region. Details of PCR amplification,
sequence of primers, cloning, and sequencing of probe are given in the
text. Primers 13a and 13b, used for PCR, are indicated by bold letters.
The right and left arrows indicate, respectively, direct and
complementary sequences of the primers given in the text. The
continuous underlining indicates the 70-bp probe sequence.
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Detection of Leishmania DNA in different biological
samples.
Over a period of 6 months, 124 dogs were tested by PCR
performed with DNA extracted from blood samples and lymph node
aspirates. All of these animals were also tested by IFAT, which yielded
42 positive, 69 negative, and 13 dubious results. This group of dogs comprised 37 animals suspected for leishmaniasis on the bases of clear
clinical signs; the others were deemed to have some risk of infection
(Table 1). We took samples of lymph node
aspirates for PCR testing, cultural isolations, and microscopic
examination from the 37 animals having clear clinical signs, and from
the 15 animals without signs that tested both positive and negative by
IFAT. Parasite isolations were obtained in 14 blood samples and in 26 lymph node aspirates. However, microscopic examination of blood and
lymph node smears detected parasites in 36 blood samples and in 47 lymph node samples (Table 2).
Representative examples of PCR products from gel electrophoresis
analysis are shown in Fig. 3. Clear
amplification signals of the 116-bp fragment in blood samples (Fig. 3A)
and lymph node aspirate samples (Fig. 3B) are detectable. The lymph
node aspirate and blood samples were taken from the same animals.
Amplification was demonstrated by comparison with the positive control
containing 1 ng of total Leishmania DNA. In lane 8 of Fig.
3A, no signal is detectable in the blood sample, whereas the amplified
fragment is present in the corresponding lymph node sample (Fig. 3B),
showing that in some cases the parasites are not detectable in blood
sample but can be present in the lymph node aspirate.
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TABLE 1.
Correlation of IFAT and PCR-dot blot test results
obtained with different kind of samples from dogs with and without
signs of leishmaniasis
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FIG. 3.
Analysis of PCR-amplified 116-bp fragment, by 2.5%
agarose gel electrophoresis. DNA was extracted from blood samples (A)
and lymph node aspirates (B). Details of PCR amplification, sequence of
primers, cloning, and sequencing of probe are given in the text.
Samples of 50 µl of PCR products were analyzed. (A) Lanes 1 to 12, 116-bp amplified fragment from blood samples. (B) Lanes 1 to 12, 116-bp
amplified fragment from lymph node aspirates. (A and B) Lane 13, control without Leishmania DNA; lane 14, positive control
amplified from 1 ng of total Leishmania DNA; lane 15, ladder
50 as a DNA molecular size marker.
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The hybridization of the 70-bp probe with amplified DNA from blood
samples and lymph node aspirates is shown in Fig.
4. Hybridization
(spots 6a and 3b)
permitted us to detect the amplification of
the 116-bp fragment in two
blood samples not clearly visible in
gel analysis (Fig.
2, lanes 6 and
9). These results show that
the dot blot hybridization can further
enhance the test sensitivity
with respect to gel electrophoresis
detection.
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FIG. 4.
Analysis of PCR products by dot blot hybridization with
11 dUTP-labeled 70-bp probe. Details of PCR amplification, sequence of
primers, cloning, and sequencing of probe are given in the text. The
samples were the same as those described in the legend to Fig. 3. Spots
1a to 6b, PCR products from blood samples; spots 1c to 6d, PCR products
from lymph node aspirate samples; C , control without
Leishmania DNA; C+, target DNA amplified from 1 ng of total
Leishmania DNA.
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The samples were deemed positive if the 116-bp fragment hybridized with
the 70-bp probe in the dot blot test. Samples were
considered negative
if no detectable PCR and dot blot hybridization
signals were found. The
dogs were considered infected when an
amplification signal was detected
in blood or lymph node aspirate
samples.
We calculated sensitivity, specificity, and positive and negative
predictive values of PCR-dot blot tests performed on the
37 animals
that had clear signs of infection and on 15 animals
without signs of
infection. For these calculations, we compared
the results obtained by
PCR on blood and lymph node aspirate samples
with those obtained by
microscopic examination, which was used
as the gold standard. The test
performed on 52 lymph node aspirates
showed sensitivity, specificity,
and positive and negative predictive
values of 100%, while values of
85, 80, 95, and 57%, respectively,
were obtained using blood samples
(Table
3).
The main goal of this study was to detect
Leishmania
infection in dogs with a test more reliable than the immunofluorescence
method. We obtained interesting results especially in the case
of
dubious reactivity to IFAT in animals suspected of having infections
or
when a negative reaction occurred in animals in the absence
or presence
of typical clinical signs. In the group of 13 dogs
with dubious IFAT
results, the PCR dot blot test was positive
in 10 blood samples and in
2 lymph node aspirates. Moreover, the
animals testing negative by IFAT
and showing the typical lesions
tested positive by PCR in 14 lymph node
aspirates and in eight
blood
samples.
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DISCUSSION |
The data presented indicate a successful attempt to satisfy the
need for a more sensitive, specific, and rapid test for the diagnosis
of leishmaniasis in dogs. We used PCR to test for Leishmania infection in dogs both with and without clinical signals of disease. Moreover, we proved that the 13a and 13b primers derived from the kDNA
constant region of L. braziliensis (27) also
amplified an L. infantum fragment inside the corresponding
kDNA region. Here, we report the sequence of the 116-bp cloned fragment
because it didn't show complete homology with all the
Leishmania kDNA-correlated sequences in the data banks.
Based on the findings of others, we supposed that a certain percentage
of sequence divergence was in the constant region among the minicircle
classes (12, 25, 29). It could arise from recombination or
mutation events occurring in the kDNA molecules (29). During
our investigation, all the isolated strains, identified by the official
method in Istituto Superiore di Sanità, were L. infantum ZMON1. Our results were in accordance with bibliographic
data describing ZMON1 as the only strain circulating in the
Mediterranean area (2, 15, 16). The amplification of the
116-bp fragment, revealed by detection with 70-bp probe, can detect as
little as 0.1 fg of DNA, corresponding to one parasite in the sample
volume examined. This extremely high sensitivity is due to the presence
in one parasite of multiple copies of kinetoplast minicircle DNA. The
greatest advantage of our test consists in the copy number of the
target repeated almost 500-fold in each parasite. In fact, 0.1 fg,
corresponding to approximately 1/500 of Leishmania total
genome, represents more or less 10 minicircles (22). It is a
valid alternative to other good reliable PCR methods based on the
amplification of the expressing gene (1). These systems in
fact amplify chromosomal sequences with lower copy numbers.
We performed PCR on samples of blood and lymph node aspirates from 124 dogs from different parts of Sicily. Higher sensitivity, specificity,
and positive and negative predictive values were found for PCR dot blot
tests performed on lymph node aspirates than for tests with blood
samples. The comparison of PCR results of both kinds of sample taken
from the same animal, together with cultural isolation and microscopic
examination, permitted us to establish that lymph node aspirates were
the best matrices for PCR diagnosis during the entire infection period
(Tables 1 and 2). Most lymph node samples examined were taken from dogs
with some signs of infection, because in these animals the examinable lymph nodes were ingrown. In this group, one dog tested negative by
microscopic examination and by PCR. In the group of dogs that appeared
healthy, PCR testing of 15 lymph node samples detected more positive
results than were obtained with blood samples, showing that this kind
of sample might also be utilized in large-scale screening.
However, blood sampling is less invasive and easily performed. Both of
these samples could be considered useful for accurate screening of
leishmaniasis in dogs with higher precision than IFAT. The comparison
between IFAT and PCR results on lymph node aspirate samples from dogs
with clinical signs permits us to deem 38% of IFAT results false
negative. It has been supposed that this highly reliable method could
be employed to make early diagnoses and to prevent the spreading of
infection. In many cases, the absence of visible lesions cannot exclude
the presence of early infection, and the IFAT result could be negative
for immunocompromised dogs because of disease. The PCR diagnosis is
independent of immune state, being a direct test that screens for the
presence of parasite DNA in the sample. During our investigation, four
dogs with positive PCR results and dubious serological results became
IFAT positive 1 month after the first test and developed the disease.
Moreover, we found only two false-positive dogs, meaning that there is
a very low probability of DNA contamination during sample manipulation. The PCR-hybridization method is rapid: it takes 48 to 72 h from sampling to the detection of the infection. It may be very useful to
carry out the PCR in parallel with the officially approved serological
test, especially in the case of dubious reactions or anergy, or in the
presence of cross-reactivity with correlated antigenic determinants.
Moreover, it may be possible to use this test in epidemiological
studies aimed to determine the prevalence in areas in which the disease
has not been controlled. Our assays give results in accordance with
those of other authors that compared PCR with the IFAT (4, 21,
26), so we conclude that the serological data alone are not
enough to make correct diagnoses of leishmaniasis and that using PCR
could help reveal further cases of infection in dogs.
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ACKNOWLEDGMENTS |
We thank M. Piazza for Leishmania strain isolation,
cultivation, and direct microscopic examination of samples.
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FOOTNOTES |
*
Corresponding author. Mailing address: Istituto
Zooprofilattico Sperimentale Della Sicilia, Via Rocco Dicillo No. 4, 90129 Palermo, Italy. Phone: (39) 91-6565234. Fax: (39) 91-6565233. E-mail: reale{at}pa.izs.it.
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Journal of Clinical Microbiology, September 1999, p. 2931-2935, Vol. 37, No. 9
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
