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Brief Report
Polymerase Chain Reaction as a
Diagnostic Tool for Detecting Leishmania
M. Brecelj, F. Pikelj, F. Gubenšek, G. Anderluh
Summary
Polymerase chain reaction (PCR) has been used to
identify a Leishmania parasite in a cutaneous ulcer from
a 27-year-old patient infected during travel in Peru. The
available classical diagnostic methods could not confirm
the diagnosis in a sufficiently short time. Therefore, two
sets of oligonucleotides were designed and with both of
them fragments of the expected size were obtained. The
sequence of the fragment derived from kinetoplast DNA
corresponds to the Leishmania Viannia complex.
Polymerase chain reaction has advantages over classical
diagnostic methods, which makes it an important
technique in those hospitals and clinical laboratories in
Europe which lack standard diagnostic tests for
Leishmania.
Key Words
Leishmania · Diagnosis · Polymerase chain reaction ·
Mini-exon · Kinetoplast DNA
Infection 2000;28:111–113
Introduction
Leishmaniasis is a parasitic disease, endemic in 88 countries
around the world. It is estimated that about 350 million people are at risk of infection and that two million new cases
of leishmaniasis occur annually. The disease has a wide
range of clinical symptoms, cutaneous, mucocutaneous and
visceral. Cutaneous leishmaniasis has been a recurrent
problem for settlers and travelers in the tropical and subtropical regions of the Old and New World. Travelers, infected during the journey, usually develop a full clinical picture within a few months after they return home, when the
disease is frequently misdiagnosed or recognized only after a delay. Furthermore, laboratories in areas where leishmaniasis is seldom encountered lack the appropriate diagnostic capabilities. Proper identification of the parasite
species is important for the treatment and prognosis of the
disease [1–3]. Standard diagnostic methods include direct
microscopy, histological examination with Giemsa staining
and immunohistochemistry, microbiological cultures and
Infection 28 · 2000 · No. 2 © URBAN & VOGEL
serological methods. In recent years the polymerase chain
reaction (PCR) has been successfully introduced and
proved to be a sensitive and powerful tool for determining
the parasite species in visceral [4–7] and cutaneous [8–10]
leishmaniasis. However, because of the lack of appropriate
oligonucleotides and commercial kits, reports of successful
diagnosis in clinical laboratories, especially in Europe, have
been scarce.
A patient with a cutaneous lesion was admitted to the
Clinic for Infectious Diseases at the Clinical Centre, Ljubljana, Slovenia, 3 months after traveling to Peru. The clinical picture suggested a diagnosis of leishmanian ulcer.
Since none of the quick diagnostic methods available could
confirm the presence of the parasites, we tried to prove the
etiology of the lesion using PCR. Two sets of oligonucleotides were used to amplify minicircle kinetoplast DNA
(kDNA) and mini-exon gene from Leishmania DNA. Both
sets of primers yielded products of the expected size. The
sequence of the minicircle fragment was determined and
confirmed the presence of kDNA of the Leishmania Viannia complex. The approach used is suitable for diagnosis in
centers not equipped for classical diagnostic evaluation of
leishmanial infections, since it is fast, easy to perform and
does not need large amounts of highly purified DNA.
Materials and Methods
A 27-year-old patient was admitted to the Clinic for Infectious Diseases, University Clinical Centre, Ljubljana, Slovenia, 3 months after returning from a 3-month visit to Peru. He presented with a cutaneous lesion on his right shank, that began as a red papule at the
M. Brecelj, F. Pikelj
Clinic for Infectious Diseases, Clinical Centre, Japljeva 2,
SLO-1000 Ljubljana, Slovenia
F. Gubenšek
Dept. of Biochemistry and Molecular Biology, Jozef Stefan Institute,
Jamova 39, SLO-1000 Ljubljana, Slovenia, and Faculty of Chemistry and
Chemical Technology, University of Ljubljana, Askerceva 5,
SLO-1001 Ljubljana, Slovenia
G. Anderluh (corresponding author)
Dept. of Biology, Biotechnical Faculty, University of Ljubljana,
Vecna pot 111, SLO-1000 Ljubljana, Slovenia; Phone: +386-61-1233388,
Fax: +386-61-273390, e-mail: [email protected]
Received: May 17, 1999 • Revision accepted: January 25, 2000
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M. Brecelj et al.: PCR detection of Leishmania
site of an insect bite and had gradually developed into a cutaneous
ulcer measuring 7 5 cm in size. The lesion had been accompanied by painful enlargement of a regional lymph node over the
previous 2 weeks. Direct examination of Giemsa-stained material,
obtained by scarification of the ulcer edge, did not lead to a diagnosis. Biopsy specimens of the ulcer edge and the enlarged lymph
node were cultivated on NNN medium for 3 weeks. The specimen
from the ulcer edge yielded a growth of leishmanial parasites,
while the lymph node specimen remained sterile. Enzyme-linked
immunosorbent assay (ELISA) using antigens of Leishmania
donovani was performed twice at an interval of 4 weeks.The serologic reactions were negative on both occasions.
Parasite DNA was isolated from the cutaneous lesion, using
SDS and proteinase K [11].A sample of approximately 5 mm3 was
dissolved in Tris-EDTA buffer, pH 8.0. After extraction with SDS
and proteinase K at 55 °C, the solution was phenolized twice and
DNA precipitated with ethanol and sodium acetate.The pellet was
resuspended in 50 µl of distilled water. In addition, a further 1-mm3
sample from the lesion was resuspended in 20 µl of 1 PCR buffer
and 3 µl was used for amplification.
Two sets of oligonucleotides were designed to amplify specifically Leishmania DNA. Oligonucleotides B1(+) and B1(–) were
used to amplify the 131-bp fragment of the conserved part of minicircle kDNA from four New World species of Leishmania [8]. The
second set of primers, composed of oligonucleotides B2(+) and
B2(–), was designed to amplify a mini-exon repeat of Leishmania
genomic DNA [9]. The sequences of the primers were as follows:
B1 (+) (5'– gga att CCC GAC ATG CCT CTG GGT A–3'), B1 (–)
(5'– cga att CAC TAT ATT ACA CCA ACC CCT A–3'), B2 (+)
(5'– gga att cAC TTT ATT GGT ATG CGA AAC TTC–3'), and
B2 (–) (5'– cga att CAG AAA CAG ATA CTT ATA TAG CG–3').
They all contained the EcoRI site (underlined). Regions of complementarity with Leishmania DNA are shown in capital letters.
For PCR amplification 50 pmoles of each oligonucleotide, 4 µl of
prepared sample and 2.5 units of Taq polymerase were used in a
total volume of 50 µl in buffer supplied by the manufacturer
(Perkin-Elmer). 30 amplification cycles were performed with denaturation at 95 °C for 1 min, annealing at 62 °C for 1 min and extension at 72 °C for 1.5 min. Products were analyzed on 1.5%
agarose gels. The fragment amplified with oligonucleotides B2(+)
and B2(–) was visible only after secondary PCR. For this, a small
portion of primary PCR mixture, usually 2 µl (1/25 of primary
PCR), was used. Fragments obtained after amplification with
B1(+) and B1(–) oligonucleotides were further digested with
EcoRI and inserted into pUC19, where their sequence was
checked using a T7 sequencing kit (Pharmacia) and S35 ∞dATP
(Amersham).
exon is composed of a moderately conserved region, consisting of an exon and an intron, from 94–140 nucleotides
in length, and a variable length spacer. Mini-exon amplification enables discrimination, according to the length of the
amplified fragment, between different Leishmania parasites: New World Dermotropic (Viannia) (approximately
230 bp), New World Dermotropic (Leishmania) (approximately 300 bp), Old World Dermotropic and Old and New
World Visceral species (approximately 410 bp). We obtained a fragment of approximately 230 bp (Figure 1) which
clearly belongs to the New World Dermotropic species of
the Viannia complex. This fragment was visible only after
the secondary PCR. This is a consequence of the relatively
low abundance of mini-exon DNA (100–200 copies per
genome) in contrast to kDNA (up to 10,000 copies per
kinetoplast), from which fragments were obtained readily
in the first round of amplification.
With the other set of oligonucleotides we amplified a
part of the conserved minicircle kDNA. kDNA has been
most often used as a source of Leishmania DNA for PCR
diagnosis. It is found exclusively within large mitochondria
called kinetoplasts and is composed of about twenty 20 kbp
large circles and up to 10,000 copies of approximately 800
bp minicircles. The minicircle DNA is a suitable target for
diagnostic PCR, due to its abundance [12]. De Bruijn and
Barker [8] sequenced minicircle from four species of Leishmania (Viannia) braziliensis complex and derived oligonucleotides for use in diagnosis of parasites from this complex.
On the basis of their data, we designed oligonucleotides
B1(+) and B1(–) which enabled amplification of a 131-bp
constant region of minicircles. The fragment obtained using these primers was of expected size. In addition, we determined the nucleotide sequence and found it to match the
published sequence of L. (V.) braziliensis complex [8].
Further, we have shown that it is not necessary to use
a common protocol of DNA isolation using proteinase K
and phenol extraction [5, 11, 13]. As is evident from figure
1 (lane 2) the fragment was also obtained by simply resuspending a few µl of scarificate from the ulcer edge in 1 PCR buffer and using such a mixture for amplification.
Results and Discussion
PCR was employed to establish the presence of Leishmania in the skin ulcer of the patient. Figure 1 shows the results of amplification using the sample of Leishmania DNA
from the patient. With both sets of oligonucleotides, fragments of the expected sizes were amplified with template
DNA extracted by standard procedures [11]. To exclude
contamination with the patient’s DNA the same conditions
were used for amplification of human DNA. In this case, no
products were observed (data not shown).
The B2(+) and B2(–) pair of oligonucleotides was designed according to the sequence of the mini-exon repeat
from the genome of Leishmania parasites [9]. The mini-
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Figure 1
Amplification of Leishmania DNA. Using B1(+) and B1(–) oligonucleotides we were able to amplify a product of 131 bp with extracted
DNA (lane 1) and DNA from the ulcer scarificate resuspended in 1 PCR buffer (lane 2). With B2(+) and B2(–) oligonucleotides the fragment of approximately 230 bp was seen only after the secondary
PCR amplification (lane 3).
M: molecular weight standards
Infection 28 · 2000 · No. 2 © URBAN & VOGEL
M. Brecelj et al.: PCR detection of Leishmania
In conclusion, PCR-based identification has proved to
be highly sensitive and easy to perform. This is particularly
important for many hospitals and clinical laboratories in
Europe, which seldom encounter cases of infection with
Leishmania, because it enables rapid confirmation of the
identity of the parasite in laboratories which are not
equipped for standard diagnostic tests.
Acknowledgments
The authors would like to thank Dr. Roger H. Pain for his critical
reading of the manuscript. This work was supported by a grant
from the Ministry of Science and Technology of Slovenia.
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