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Journal of Clinical Microbiology, March 2001, p. 849-854, Vol. 39, No. 3
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.3.849-854.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Development of a Species-Specific PCR Assay for
Detection of Leishmania donovani in Clinical Samples from
Patients with Kala-Azar and Post-Kala-Azar Dermal
Leishmaniasis
Poonam
Salotra,1,*
G.
Sreenivas,1
Gregory P.
Pogue,2,
Nancy
Lee,2
Hira L.
Nakhasi,2
V.
Ramesh,3 and
N.
S.
Negi4
Institute of Pathology (ICMR), Safdarjung Hospital
Campus,1 and Departments of
Dermatology3 and
Medicine,4 Safdarjung Hospital, New
Delhi 110 029, India, and Division of Emerging and Transfusion
Transmitted Diseases, Center for Biologics Evaluation and Research,
Food and Drug Administration, Rockville, Maryland
208522
Received 2 August 2000/Returned for modification 9 October
2000/Accepted 22 December 2000
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ABSTRACT |
We have developed a PCR assay that is capable of amplifying
kinetoplast DNA (kDNA) of Leishmania donovani in a
species-specific manner among Old World leishmanias. With Indian
strains and isolates of L. donovani the assay was
sensitive enough to detect kDNA in an amount equivalent to a single
parasite or less. The extreme sensitivity of the assay was reflected in
its ability to detect parasite DNA from small volumes of peripheral
blood of patients with kala-azar (KA) and from skin lesions from
patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%)
were positive by PCR. The test provided a diagnosis of KA with 96%
sensitivity using patient whole-blood samples instead of bone marrow or
spleen aspirates that are obtained by invasive procedures. The assay
was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%).
Cross-reactions with pathogens prevalent in the area of endemicity,
viz., Mycobacterium tuberculosis, Mycobacterium leprae, and
Plasmodium spp., could be ruled out. Eighty-one control
samples, including dermal scrapings from healthy portions of skin from
patients with PKDL were all negative. Two of twenty controls from the
area of endemicity were found positive by PCR assay; however, there was
a good possibility that these two were asymptomatic carriers since they
were serologically positive for KA. Thus, this PCR assay represents a
tool for the diagnosis of KA and PKDL in Indian patients in a
noninvasive manner, with simultaneous species identification of
parasites in clinical samples.
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INTRODUCTION |
The protozoan parasites of the genus
Leishmania are the causative agents of a group of diseases
called leishmaniases, endemic in more than 80 countries worldwide.
Considerable morbidity and mortality occur in the visceral infections
termed visceral leishmaniasis (VL) or kala-azar (KA), which is a
significant infectious disease in the developing world and of late in
the developed world because of increased international travel and human
immunodeficiency virus (HIV) infection. KA is a symptomatic infection
of the liver, spleen, and bone marrow caused by organisms of
Leishmania donovani complex. The annual incidence and
prevalence of VL cases worldwide are 0.5 million and 2.5 million
respectively. Of these 90% of cases occur in India, Nepal, Bangladesh,
and Sudan (9). The official estimate of 430,000 VL cases
in Bihar State of India over the past 11 years may represent only a
fraction of the real numbers. The actual number is believed to be at
least five times as great (8). The causative organism in
the Indian subcontinent and Africa is L. donovani donovani,
while in the Mediterranean basin and South America it is L. donovani infantum.
Post-KA dermal leishmaniasis (PKDL) is an unusual dermatosis that
develops as a sequel of KA, producing gross cutaneous lesions in the
form of hypopigmented macules, erythema, and nodules. The disease is
relatively common in the Indian subcontinent and less frequent in East
Africa but exceptional in the American and European continents
(25). The need to search for cases of PKDL and treat them
as a part of KA control programs has been emphasized since patients
with PKDL provide the only known reservoir for the parasite in India
(37). The cost and toxicity of current treatment regimens emphasize the importance of establishing control strategies and make
diagnosis and typing of leishmaniasis critical (20).
Detection and characterization of Leishmania from patients
with KA or PKDL are important for deciding treatment regimens as well
as for understanding the disease epidemiology. Current diagnostic
methods based on parasite detection (stained smears, culture, and
histopathology) and immunological methods (direct agglutination test,
enzyme-linked immunosorbent assay [ELISA], etc.) have several
limitations, including low sensitivity and specificity. Procedures for
demonstration of the parasite in spleen or bone marrow in KA and in
skin lesions in PKDL are invasive and often not sensitive enough.
Immunological methods fail to distinguish between past and present
infections and are not reliable in the case of immunocompromised
patients (6, 11, 39). Furthermore, neither of
these methods addresses the problem of species identification, which is
important for determining appropriate treatment regimens and designing
control measures. Procedures involving the use of monoclonal
antibodies, isoenzyme and schizodeme analysis, and DNA hybridization
have to be resorted to (12, 17, 29). Most of these
procedures are tedious and require massive cultures of parasites. There
is, therefore, an urgent need to develop diagnostic procedures that are
simple, sensitive, and specific.
In recent years PCR-based diagnostic methods have been described for
leishmaniasis, with a wide range of sensitivity and specificity. An
excellent target for a sensitive and rapid detection method is the
kinetoplast mini-circle DNA, which is present at thousands of copies
per cell. The mini-circles have been used as targets for selective
amplification of parasite DNA in various studies (5, 7, 21, 28,
35). The identification of conserved sequence elements
represented within the kinetoplast DNA (kDNA) of a given species of
Leishmania would allow the design of oligonucleotide primers
to be used for species-specific identification of parasites in clinical
samples. We have analyzed kDNA sequences from Old World leishmanias and
designed primers specific for L. donovani species to detect
kDNA from a single parasite in the presence of huge excesses of human
DNA. The utility of the primers designed for L. donovani has
been examined in clinical samples from patients with KA and PKDL in
India. The PCR test was found to be sensitive enough to detect parasite
DNA from peripheral blood of patients with KA and from skin lesions of
patients with PKDL. Furthermore, the test was specific for L. donovani species of the parasite, leading to simultaneous species
identification of the parasite.
| |
MATERIALS AND METHODS |
Patients.
Fifty-one KA patients hailing from Bihar and
reporting to Safdarjung Hospital (SJH), New Delhi, India, were included
in the study at the pretreatment stage. The patients presented with
characteristic symptoms of KA such as fever, hepatosplenomegaly,
anemia, and leukopenia. Only those patients for whom the diagnosis of
KA was confirmed by demonstration of parasites in bone marrow aspirates were taken for the study. Blood was taken from all 51 patients. In
addition bone marrow samples were obtained from 8 of these patients.
Clinical samples were also taken from a total of 48 patients with PKDL
that were originally from Bihar and reported to the Dermatology
Department of SJH during the period from 1996 to 2000. Forty-five of
these reported history of KA, while the remaining three were not aware
of it. The time elapsed after cure from KA in the 45 patients ranged
from 1 to 15 years. Clinical diagnosis in 36 patients was based on
condition characterized by erythematous indurate areas and
papulonodular and hypochromic macules in a bilateral distribution. The
remaining 12 patients had a predominantly macular presentation, most of
them being the subject of a recent study (26). Slit skin
smears stained with Giemsa stain were positive in only 10 cases.
Histopathological findings upon skin biopsy were similar to those
reported earlier (19, 31). The dermis showed a diffuse
infiltration by lymphocytes, histiocytes, and plasma cells. All
patients responded well to therapy with sodium antimony gluconate. The
control group of patients was comprised of individuals with confirmed
cases of malaria (n = 15), pulmonary tuberculosis (n = 15), and lepromatous leprosy (n = 32) from SJH. Twenty
healthy volunteers living in an area of endemicity (Muzaffarpur, Bihar)
were also included in the control group.
Parasites.
Ten World Health Organization reference strains
of Leishmania originating from distinct geographic locations
were used in the study. These included L. donovani DD8
(MHOM/IN/80/DD8) from India, L. donovani AG83
(MHOM/IN/83/AG83) from India, L. donovani 1S
(MHOM/SD/00/1S-C12D) from Sudan, L. donovani WR 684 (MHOM/ET/67/82) from Ethiopia, L. donovani infantum
(MCAN/SP/00/XXX) from Spain, L. tropica WR 683 (MHOM/SU/58/OD) from the Soviet Union, L. tropica WR
664 (MHOM/SU/74/K27) from the Soviet Union, L. major WR662 (MHOM/IL/67/Zericho II/WR662) from Israel, L. major
LV39 (MRHO/SU/59/P/LV39) from the Soviet Union, and L. major
ASKH (MHOM/SU/73/5ASKH) from the Soviet Union. Three isolates of
L. donovani (MHOM/IN/94/IICB6, MHOM/IN/94/IICB7, and
MHOM/IN/94/IICB8) were kindly provided by D. Sarkar, Indian Institute
of Chemical Biology, Calcutta, India. These were isolated from patients
with VL (IICB6 and IICB8) and PKDL (IICB7) originating from Bihar and
characterized as L. donovani (10). Ten parasite
isolates were set up in culture in our laboratory over the last 2 years
from patients with VL and PKDL reporting to SJH. All parasite cultures
were set up and propagated in medium 199 supplemented with 25 mM HEPES,
pH 7.5, and 10% fetal calf serum (30). Parasites were
harvested in late log phase and washed in phosphate-buffered saline
prior to DNA isolation.
Sample collection and DNA isolation.
Bone marrow and skin
scrapings were collected in NET buffer (150 mM NaCl, 15 mM Tris-HCl
[pH 8.30], 1 mM EDTA). Blood was collected in heparinized tubes.
Samples were transported to the laboratory at ambient temperature,
except for blood collected in areas of endemicity, in which case they
were brought on ice. Samples were transferred to 4°C and generally
processed on the same day. Blood (0.2 to 1 ml) was treated with RBC
lysis buffer (114 mM sodium phosphate [pH 8.0], 1 mM
NH4Cl), and the buffycoat was isolated. DNA from parasite
cultures as well as from clinical samples (skin scrapings, bone marrow,
or blood) was isolated by overnight lysis in NET buffer with
proteinase-K (100 µg/ml) and 1% sodium dodecyl sulfate. DNA was
extracted by phenol-chloroform extraction and ethanol precipitation. In
a few samples DNA was isolated from 0.2 ml of blood using a QIAamp DNA
blood minikit (Qiagen) in order to determine if this method provided
any advantage over the phenol-chloroform method for DNA extraction.
Oligonucleotide primers.
The 792-bp L. donovani
kinetoplast mini-circle sequence (accession no. Y11401) was analyzed
using PC-Gene software, and appropriate primers were identified. The
two primers used were 5'-AAATCGGC TCCGAGGCGGGAAAC-3' and 5'-GGTACACTCTATCAGTAGCAC-3', together
designated the LdI primers. These were synthesized using an Applied
Biosystems DNA-RNA synthesizer (model 394). The LdI primers amplify a
fragment of approximately 600 bp that is seen on the gels.
PCR amplification.
DNA from cultured parasites (1 ng) and
from clinical samples (100 ng) was taken for amplification using the
LdI primers described above. The reaction mixture (50 µl) contained
10 mM Tris-HCl (pH 8.3) 50 mM KCl, 1.5 mM MgCl2, a 200 µM
concentration of each deoxynucleoside triphosphate, 50 ng of each
primer, and 1.25 U of Taq DNA polymerase (Gibco BRL). Each
reaction mixture was overlaid with mineral oil, and amplification was
performed in a thermal cycler (Perkin-Elmer, Warrington, Great Britain)
programmed for 40 cycles of denaturation at 94°C for 1 min, annealing
at 45°C for 1 min, and extension at 72°C for 2 min, preceded by an
initial denaturation of 2 min at 94°C. Final extension was for 3 min at 72°C. Products were analyzed by electrophoresis in 1% agarose
gel containing ethidium bromide (0.5 µg/ml) in TAE buffer (0.04 M
Tris acetate, 0.001 M EDTA) and photographed under UV illumination.
Southern blot analysis.
PCR products were analyzed in 1%
agarose gel, and Southern blot analysis was done as described
(14). Southern blots were hybridized with
32P-labeled cloned L. donovani kDNA fragments
using the conditions described (14).
Sequencing reaction.
The PCR amplification products from
culture isolates and clinical samples from patients with KA and PKDL
were cloned into the pGEMT-Easy vector system (Promega). DNA sequencing
was performed with the ABI PRISM Dye Terminator Cycle sequencing kit
and an ABI PRISM automated sequencer (Model 377; Perkin-Elmer).
Briefly, the sequencing reaction mixture contained terminator ready
reaction mix, DNA template, primer and 5% dimethyl sulfoxide. Dimethyl sulfoxide was added to keep the DNA template denatured since leishmania DNA has a high GC content. The PCR was carried out in a DNA thermal cycler (model 480). The PCR conditions in the Perkin-Elmer analytical manual were followed. Sequences were assembled and edited using Sequencher software (Gene Codes Corporation, Ann Arbor, Mich.) and
analyzed with Mac Vector DNA and protein sequence analysis software
(Genetics Computer Group Inc., Madison, Wis.).
| |
RESULTS |
The initial aim of the study was to define a set of PCR primers
based on kDNA sequences which would allow sensitive and specific detection of L. donovani. For this purpose, we analyzed kDNA
mini-circle sequences from an L. donovani DD8 strain of
Indian origin and designed oligonucleotide primers that showed lack of
cross-reactivity with organisms phylogenetically or geographically
related (G. P. Pogue and H. L. Nakhasi, unpublished data).
The sensitivity and effectiveness of the PCR-based detection system was
seen in its ability to amplify kDNA fragments from as little as 1 fg of DNA of L. donovani (Fig. 1).
When the amplification properties of PCR were combined with the
specificity and sensitivity of Southern blot-based DNA
hybridization, kDNA fragments could be detected by probes generated
from the parasite kDNA sequences in PCRs containing as little as 10 fg
of Leishmania DNA diluted in a 10 million-fold excess of
human DNA (Fig. 2).
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FIG. 1.
Sensitivity of the PCR assay. Shown are the results of
PCR amplification of the serially diluted L. donovani
(DD8) DNA analyzed on agarose gels. DNA was extracted from parasite
cultures and amplified as described in Materials and Methods. Lane M, 1 kb Ladder (Gibco BRL); lane 1, 10 ng of DNA; lane 2, 1 ng of DNA;
lane 3, 10 pg of DNA; lane 4, 1 pg of DNA; lane 5, 10 fg of DNA; lane
6, 1 fg of DNA.
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FIG. 2.
Sensitivity of PCR amplification of
Leishmania kDNA followed by Southern blot analysis. The PCR
contained 100 ng of human genomic DNA and the indicated amount of total
DNA from L. donovani DD8. The PCR product was probed
with parasite kDNA and exposed for about 1 h. Lane 4 represents a
PCR containing only human DNA as a control.
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Initially the primers were evaluated with various strains of Old
World Leishmania as described under Materials and
Methods. Both strains of L. donovani of
Indian origin gave positive results by PCR, as did the three isolates
from Indian patients (Fig. 3, lanes 1 to
5). These three isolates of L. donovani were isolated 6 years prior to the present study from patients with KA and PKDL and
were preserved in the Parasite Bank at the IICB. Strains of L. donovani from Sudan and Ethiopia as well as
L. donovani infantum from Spain were positive by
PCR, though the bands were of significantly lower intensity
(Fig. 3, lanes 6 to 8). DNA from L. major and L. tropica was not amplified, indicating the
species specificity of primers (Fig. 3, lanes 9 and 10). Species
specificity for L. donovani was further established,
since the use of DNA up to 10 ng from three different strains of
L. major and two strains of L. tropica
as described in Materials and Methods did not give any amplification.
Specificity of the primers was also evaluated using DNA (10 ng) from
microorganisms causative of the common infectious diseases prevalent in
India, such as Plasmodium spp., Mycobacterium leprae,
Mycobacterium tuberculosis; there was no amplification with DNA
from any of these organisms using our primers (Fig. 3, lanes 11 to 13).
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FIG. 3.
Amplification of parasite DNA from various strains and
isolates of Leishmania. DNA (1 ng) isolated from parasite
cultures was subjected to PCR and analyzed. Lane 1, L. donovani AG83; lane 2, L. donovani DD8; lane 3, L. donovani IICB8; lane 4, L. donovani
IICB6; lane 5, L. donovani IICB 7 (PKDL origin); lane
6, L. donovani 1S; lane 7, L. donovani
WR684; lane 8 L. donovani infantum; lane 9, L. tropica WR683; lane 10, L. major LV
39, lane M, 1-kb ladder, lane 11, Plasmodium; lane 12, M. leprae; lane 13, M. tuberculosis.
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In order to establish the clinical utility of the assay, PCR
amplification was evaluated with DNA from several recent isolates of
the parasite. Parasite cultures were set up from bone marrow aspirates
of five patients with KA that reported to SJH over the last 2 years
(designated KA1 to KA5). DNA isolated from each of these cultures was
observed to be amplified by PCR (Fig. 4,
lanes 1 to 5). The assay was also positive with a number of cultures isolated from dermal lesions of patients with PKDL (PK1 to PK5) (Fig.
4, lanes 6 to 10), while the parasite culture isolated from a patient
with cutaneous leishmaniasis hailing from Afghanistan gave no
amplification in the PCR test (Fig. 4, lane 11). The sensitivity of the assay with the isolates of KA and PKDL was found to be 1 fg of
total DNA.
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FIG. 4.
DNA amplification from recent field isolates of KA and
PKDL. DNA (1 ng) extracted from cultures of parasite isolates was
used for PCR amplification. Lanes: M, 1-kb ladder; 1, KA-1; 2, KA-2; 3, KA-3; 4, KA-4; 5, KA-5; 6, PK-1; 7, PK-2; 8, PK-3; 9, PK-4; 10, PK-5;
11, isolate from a patient with cutaneous leishmaniasis.
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A clinical study was undertaken with Indian patients with KA and PKDL
using PCR based on LdI primers. The PCR assay was evaluated with
clinical samples from patients with KA and PKDL along with samples from
suitable controls. PCR analysis of representative sample from each of
test materials
i.e., bone marrow and whole blood from patients with
KA, blood from patients with malaria and tuberculosis, blood from
control subjects from areas of endemicity, and skin lesion samples from
patients with PKDL and leprosyis shown in Fig.
5. Only samples of bone marrow and blood
from patients with KA and from PKDL skin lesion specimens were PCR
positive (Fig. 5, lanes 1, 2, and 6). The rest of the samples were
negative (Fig. 5, lanes 3 to 5 and 7). All eight samples of bone marrow aspirates of patients with KA gave positive results when subjected to
PCR amplification (Table 1). Our results
showed that the primers could specifically amplify DNA from peripheral
blood of 49 of 51 patients with KA. Identical results were obtained by
PCR using DNA extracted by the phenol-chloroform method or with a
QIAamp DNA blood minikit, indicating that either method could be
employed. DNA from just 0.2 ml of patient's blood was found to be
sufficient for the PCR test, indicating the tremendous clinical
usefulness of the test. All malaria (n = 15) and
tuberculosis (n = 15) blood samples were negative, while
two of the 20 samples from control subjects from areas of endemicity
were positive by PCR (Table 1). A large majority of specimens from
patients with PKDL (45 of 48) gave positive results, while all the
specimens from patients with leprosy (32 of 32) were negative.
Samples of normal dermal tissue from unaffected parts of skin of
patients with PKDL (n = 19) were also negative (Table 1).
Sequence analysis of the PCR product obtained with DNA from clinical
samples (for KA, blood; for PKDL, tissue) as well as from parasite
isolates from patients with KA and PKDL revealed that the sequence of
the products was identical to that obtained with the DD8 strain of
L. donovani (Fig. 6).
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FIG. 5.
PCR assay with clinical samples of KA and PKDL. DNA
(100 ng) isolated from clinical samples was used for PCR amplification.
Lane M, 1-kb ladder, lane 1, KA (bone marrow); lane 2, KA (blood); lane
3, malaria (blood); lane 4, tuberculosis (blood); lane 5, control from
the area of endemicity (blood); lane 6, PKDL (skin lesion); lane
7, leprosy (lesion).
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FIG. 6.
Sequence of PCR product with DNA isolated from
L. donovani DD8 strain and isolates and clinical
samples from patients with KA and PKDL. PCR products obtained with
DNA isolated from L. donovani DD8 strain, parasite
isolates from patients with KA and PKDL (two each) and clinical samples
(two each of KA blood and PKDL tissue) were subjected to sequence
analysis. Identical sequences were obtained for the PCR products in
each case, which matched exactly with the published sequence of a
792-bp kDNA mini-circle segment of the DD8 strain of L. donovani (GenBank accession no. Y11401). The positions of primers
are indicated in boldface type.
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DISCUSSION |
We have developed a PCR assay that is species specific for
L. donovani kDNA among the Old World leishmanias and
could detect the parasite in a highly sensitive manner in clinical
samples from Indian patients with KA and PKDL. The assay could
detect as little as 1 fg of parasite DNA from Indian strains of
L. donovani, an amount that represents the equivalent
of approximately 0.1 parasite (13). DNAs from several
parasite isolates obtained from patients with KA as well as PKDL
originating from the region of endemicity in India were found to be
amplified with equal sensitivity. Therefore, the assay is theoretically
capable of detecting a single parasite in a biological sample.
The extreme sensitivity of our detection system was evident by
its ability to amplify parasite DNA from peripheral blood of patients
with KA and dermal lesions of patients with PKDL in a large majority of
cases. A total of 107 clinical samples from patients with leishmaniasis
were examined, and 95% tested positive by PCR. Our PCR described in
this work yielded a unique product of approximately 600 bp, and no
nonspecific side product or artifacts appeared on the gel. It had the
advantage that results were easily and unequivocally
interpreted upon analysis of agarose gels. The high level of
sensitivity was reflected by the ability of the assay to detect
parasite DNA in peripheral blood of patients with KA with 96%
sensitivity in the 51 cases examined. Use of peripheral blood is
advantageous because the collection procedure is less invasive and
safer than the splenic or bone marrow biopsy specimen collection. In
earlier studies for diagnosis of VL due to L. donovani,
the sensitivity of PCR for blood samples has been found to be in the
range of 45 to 94% based on smaller sample sizes ranging from 17 to 42 (1, 4, 15, 21, 22, 32, 35). For detection of VL due to
L. donovani infantum, which may have a different
pathogenesis, sensitivities between 64 to 97% have been reported with
blood samples (16, 18, 21). The sensitivity of detection
was 100% in the limited number of bone marrow samples that we
examined. Bone marrow is known to have a high load of parasites, while
in peripheral blood the parasites are relatively scarce. Studies
reporting PCRs with detection sensitivities comparable to ours (less
than a single parasite) did not obtain sensitivities as high as that of
our assay when using blood samples of patients with KA (15,
35). With clinical samples the sensitivity in practice may be
affected by factors such as accessibility of the DNA in
parasite-containing biopsy samples and the conditions used in the PCR amplification.
DNA isolated from the pathogens causative of common coendemic diseases
(M. leprae, M. tuberculosis, and Plasmodium) was
not amplified. Blood from patients with malaria and tuberculosis were PCR negative in all cases (30 of 30), while two of the blood samples from control subjects from areas of endemicity were PCR positive, giving an overall specificity of 96% in the control blood samples examined. The two positive controls from areas of endemicity were relatives of patients with KA and possibly asymptomatic carriers since
both samples were positive by ELISA with recombinant antigen k39 and in
a dipstick test using immunochromatographic strips coated with rk39
antigen (P. Salotra and G. Sreenivas, unpublished data), tests reported
to be specific for KA (34, 36). A recent study has
reported a PCR assay that could often detect parasitemia a few weeks
before the appearance of any clinical signs or symptoms (16).
In India, 10 to 20% percent of patients apparently cured of KA develop
PKDL. As there is no known animal reservoir in India, patients
with PKDL are considered an important source of transmission in recent
epidemics of KA in India (37). The disease is easily confused with a number of skin disorders, primarily leprosy, due to
similarities in the clinical presentation, therefore, a high level of
clinical expertise is needed to diagnose PKDL. Detection of
L. donovani bodies in skin lesions by microscopy gives
a positive result in only about 58% of cases, as parasites are scanty
(33). Early recognition and treatment of PKDL would
contribute significantly to the control of KA, as patients with PKDL
constitute a reservoir for the Leishmania parasite
(37, 38). Our assay, validated in a large number of
cases, provided a highly sensitive method for diagnosis of PKDL.
The sensitivity of the assay was 93.8% for PKDL, which is
significantly higher than that reported (82.7%) in a recent study
with 32 patients with PKDL in Sudan (23). The
specificity of the test was 100%, as all of the control tissues examined (32 leprosy lesions and 19 dermal samples from healthy regions
of skin of patients with PKDL were negative.
Species specificity of the assay was carefully evaluated by testing DNA
from different strains and species of Old World Leishmania. The assay was found to be positive with several World Health
Organization reference strains of L. donovani
originating from distinct geographical regions. L. donovani from Ethiopia and Sudan and L. donovani
infantum from Spain gave PCR products of identical size but
of comparatively lower intensity, probably due to lower copy numbers of
the target kDNA sequence. Variations among L. donovani
strains from different geographic regions have also been detected by
random amplified polymorphic DNA PCR (3) and arbitrary
primed PCR analysis (24). The primers were found to
be species specific for L. donovani, as DNA
from two other Leishmania species examined (L. major and L. tropica) was not amplified. One
clinical isolate of L. tropica from a patient with
cutaneous leishmaniasis was also negative, while several clinical
isolates from patients with KA and PKDL were all positive. The PCR
products amplified from clinical samples of patients with KA and PKDL
showed nucleotide sequences identical to those of the cultured parasites.
Our PCR provides a useful tool for the simultaneous typing of parasites
while diagnosis is being performed on clinical samples. Such a tool is
necessary to complement diagnostic assays since most of them do not
furnish the taxonomic information about the parasite required to
determine the appropriate therapeutic regimens and control measures.
Early detection and simultaneous typing would enable implementation of
specific treatment. Leishmania is increasingly recognized as
an opportunistic pathogen during coinfection with HIV (2,
27). Since incidence of HIV infection is on the increase in
India, cases of coinfection with Leishmania are likely to
present in future. In such cases immunological tests have particularly
low sensitivity, and our assay would provide a rapid detection as well
as species identification of Leishmania. Since this method
is rapid and reproducible, we believe that it can be used for the
reliable identification and characterization of cultured parasites. The
test has other potential values in detecting and typing parasites
in vectors for epidemiological surveys and in retrospective
studies of archival material.
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ACKNOWLEDGMENTS |
We thank Shyam Sundar, Banaras Hindu University, Varanasi, India,
for helping us procure blood samples from controls in areas of
endemicity and Dwijen Sarkar, IICB, for providing strains and isolates
of Leishmania. We acknowledge the expert technical
assistance of P. D. Sharma.
Part of this work was supported by a grant from the Indo-U.S. Vaccine
Action Program.
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FOOTNOTES |
*
Corresponding author. Mailing address: Molecular
Biology Lab, Institute of Pathology (ICMR), Safdarjung Hospital Campus,
Post Box #4909, New Delhi 110 029, India. Phone: 91-11-6198402. Fax: 91-11-6198401. E-mail: salotra{at}vsnl.com.
Present address: Director, Large Scale Biology Corp.,
Vacaville, CA 95688.
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REFERENCES |
| 1.
|
Adhya, S.,
M. Chatterjee,
M. Q. Hassan,
S. Mukherjee, and S. Sen.
1995.
Detection of Leishmania in the blood of early kala-azar patients with the aid of polymerase chain reaction.
Trans. R. Soc. Trop. Med. Hyg.
89:622-624[CrossRef][Medline].
|
| 2.
|
Albrecht, H.,
I. Sobottka,
C. Emminger,
H. Jablonowski,
G. Just,
A. Stoehr,
T. Kubin,
B. Salzberger,
T. Lutz, and J. V. Lunzen.
1996.
Leishmaniasis and HIV.
Arch. Pathol. Lab. Med.
120:189-198[Medline].
|
| 3.
|
Andresen, K.,
A. Gaafar,
A. M. El Hassan,
A. Ismail,
M. Dafalla,
T. G. Theander, and A. Kharazmi.
1996.
Evaluation of the polymerase chain reaction in the diagnosis of cutaneous leishmaniasis due to Leishmania major. A comparison with direct microscopy of smears and sections from lesions.
Trans. R. Soc. Trop. Med. Hyg.
90:133-135[CrossRef][Medline].
|
| 4.
|
Andresen, K.,
S. Gasim,
A. M. El Hassan,
E. A. Khalil,
D. C. Barker,
T. G. Theander, and A. Kharazmi.
1997.
Diagnosis of visceral leishmaniasis by polymerase chain reaction using blood, bone marrow and lymph node samples from patients from the Sudan.
Trop. Med. Int. Health
2:440-444[CrossRef][Medline].
|
| 5.
|
Aviles, H.,
A. Belli,
R. Armijos,
F. P. Monroy, and E. Harris.
1999.
PCR detection and identification of Leishmania parasites in clinical specimens in Ecuador: a comparison with classical diagnostic methods.
J. Parasitol.
85:181-187[CrossRef][Medline].
|
| 6.
|
Badaro, R.,
D. Benson,
M. C. Eulalio,
M. Freire,
S. Cunha,
E. M. Netto,
D. P. Sampaio,
C. Madureira,
J. M. Burns,
R. L. Houghton,
J. R. David, and S. G. Reed.
1996.
rK39: A cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis.
J. Infect. Dis.
173:758-761[Medline].
|
| 7.
|
Bhattacharyya, R.,
K. Das,
S. Sen,
S. Roy, and H. K. Majumder.
1996.
Development of a genus specific primer set for detection of Leishmania parasites by polymerase chain reaction.
FEMS. Microbiol. Lett.
135:195-200[CrossRef][Medline].
|
| 8.
|
Bihar Directorate of Health Service.
1998.
Monograph on Kala-azar incidence in Bihar (1985-1991; 1991-1996).
Office of the Chief Malaria Officer, Bihar Directorate of Health Services, Patna, Bihar, India.
|
| 9.
|
Bora, D.
1999.
Epidemiology of visceral leishmaniasis in India.
Natl. Med. J. India
12:62-68[Medline].
|
| 10.
|
Chatterjee, M.,
M. Manna,
A. N. Bhaduri, and D. Sarkar.
1995.
Recent kala-azar cases in India: isozyme profiles of Leishmania parasites.
Indian J. Med. Res.
102:165-172[Medline].
|
| 11.
|
Gradoni, L.,
A. Scalone, and M. Gramiccia.
1993.
HIV-Leishmania co-infections in Italy; serological data as an indication of the sequence of acquisition of the two infections.
Trans. R. Soc. Trop. Med. Hyg.
87:94-96[CrossRef][Medline].
|
| 12.
|
Grimaldi, G., Jr.,
J. R. David, and D. MacMohan-Pratt.
1987.
Identification and distribution of New World Leishmania species characterized by serodeme analysis using monoclonal antibodies.
Am. J. Trop. Med. Hyg.
36:280-287.
|
| 13.
|
Grimaldi, G., Jr., and R. B. Tesh.
1993.
Leishmaniasis of the New World: current concepts and implications for future research.
Clin. Microbiol. Rev.
6:230-250[Abstract/Free Full Text].
|
| 14.
| Joshi, M., D. M. Dwyer, and H. L. Nakhasi. Cloning and characterization of differentially expressed
genes from in vitro grown amastigotes of Leishmania
donovani. Mol. Biochem. Parasitol. 58:345-354.
|
| 15.
|
Katakura, K.,
S.-I. Kawazu,
T. Naya,
K. Nagakura,
M. Ito,
M. Aikawa,
J.-Q. Qu,
L.-R. Guan,
X.-P. Zuo,
J.-J. Chai,
K.-P. Chang, and Y. Matsumoto.
1998.
Diagnosis of kala-azar by nested PCR based on amplification of the Leishmania mini-exon gene.
J. Clin. Microbiol.
36:2173-2177[Abstract/Free Full Text].
|
| 16.
|
Lachaud, L.,
J. Dereure,
E. Chabbert,
J. Reynes,
J. M. Mauboussin,
E. Oziol,
J. P. Dedet, and P. Bastien.
2000.
Optimized PCR using patient blood samples for diagnosis and follow-up of visceral leishmaniasis, with special reference to AIDS patients.
J. Clin. Microbiol.
38:236-240[Abstract/Free Full Text].
|
| 17.
|
Lopes, U.,
H. Mohen,
G. Grimaldi, Jr.,
M. Marzachi,
R. Pacheco, and C. Morel.
1984.
Schizodeme and zymodeme characterization of Leishmania in the investigation of foci of visceral and cutaneous leishmaniasis.
J. Parasitol.
70:89-98[CrossRef][Medline].
|
| 18.
|
Mathis, A., and P. Deplazes.
1995.
PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs.
J. Clin. Microbiol.
33:1145-1149[Abstract].
|
| 19.
|
Mukherjee, A.,
V. Ramesh, and R. S. Misra.
1993.
Post kala-azar dermal leishmaniasis: a light and electron microscopic study of 18 cases.
J. Cutan. Pathol.
20:320-325[CrossRef][Medline].
|
| 20.
|
Navin, T. R.,
B. A. Arana,
F. E. Arana,
A. M. De Merida,
A. L. Castillo, and J. L. Pozuelos.
1990.
Placebo controlled clinical trials of meglumine antomoniate (glucantime) vs. localized controlled heat in the treatment of cutaneous leishmaniasis in Guatemala.
Am. J. Trop. Med. Hyg.
42:43-50.
|
| 21.
|
Nuzum, E.,
F. White III,
C. Thakur,
R. Dietze,
J. Wages,
M. Grogl, and J. Berman.
1995.
Diagnosis of symptomatic visceral leishmaniasis by use of the polymerase chain reaction on patient blood.
J. Infect. Dis.
171:751-754[Medline].
|
| 22.
|
Osman, O. F.,
L. Oskam,
E. E. Zijlstra,
N. C. Kroon,
G. J. Schoone,
E. A. G. Khalil,
A. M. Hassan, and P. A. Karger.
1997.
Evaluation of PCR for diagnosis of visceral leishmaniasis.
J. Clin. Microbiol.
35:2454-2457[Abstract].
|
| 23.
|
Osman, O. F.,
L. Oskam,
N. C. M. Kroon,
G. J. Schoone,
E.-T. A. G. Khalil,
A. M. El Hassan,
E. E. Zijlstra, and P. A. Kager.
1998.
Use of PCR for diagnosis of post-kala-azar dermal leishmaniasis.
J. Clin. Microbiol.
36:1621-1624[Abstract/Free Full Text].
|
| 24.
|
Pogue, G. P.,
S. Koul,
N. S. Lee,
D. M. Dwyer, and H. L. Nakhasi.
1995.
Identification of intra- and interspecific Leishmania genetic polymorphisms by arbitrary primed polymerase chain reactions and use of polymorphic DNA to identify differentially regulated genes.
Parasitol. Res.
81:282-290[CrossRef][Medline].
|
| 25.
|
Ramesh, V., and A. Mukherjee.
1995.
Post kala-azar dermal leishmaniasis.
Int. J. Dermatol.
34:85-91[Medline].
|
| 26.
|
Ramesh, V., and N. Singh.
1999.
A clinical and histopathological study of macular type of post kala-azar dermal leishmaniasis.
Trop. Doc.
29:205-207[Medline].
|
| 27.
|
Rios-Buceta, L.,
G. F. Buezo,
P. F. Penas,
E. D. Tello,
M. A. Montanes,
J. F. Fernandez, and A. G. Diez.
1996.
Post kala-azar dermal leishmaniasis in a HIV-patient.
Int. J. Dermatol.
35:303-304[Medline].
|
| 28.
|
Rodgers, M. R.,
S. J. Popper, and D. F. Wirth.
1990.
Amplification of kinetoplast as a tool in the detection and diagnosis of Leishmania.
Exp. Parasitol.
71:267-275[CrossRef][Medline].
|
| 29.
|
Rodriguez, N.,
B. Guzman,
A. Rodas,
H. Takiff,
B. Bloom, and J. Convit.
1994.
Diagnosis of cutaneous leishmaniasis and species discrimination of parasites by PCR and hybridization.
J. Clin. Microbiol.
32:2246-2251[Abstract/Free Full Text].
|
| 30.
|
Salotra, P.,
A. Raina, and N. S. Negi.
1999.
Immunoblot analysis of the antibody response to antigens of Leishmania donovani in Indian Kala-azar.
Br. J. Biomed. Sci.
56:263-267[Medline].
|
| 31.
|
Salotra, P.,
A. Raina, and V. Ramesh.
1999.
Western blot analysis of humoral immune response to Leishmania donovani in patients with post kala-azar dermal leishmaniasis.
Trans. R. Soc. Trop. Med. Hyg.
93:98-101[CrossRef][Medline].
|
| 32.
|
Singh, N.,
M. D. Curran,
A. K. Rastogi,
D. Middleton, and S. Sundar.
1999.
Diagnostic PCR with Leishmania donovani using sequences from the variable region of kinetoplast minicircle DNA.
Trop. Med. Int. Health
4:448-453[CrossRef][Medline].
|
| 33.
|
Singh, R. P.
1968.
Observations on dermal leishmanoid in Bihar.
Indian J. Dermatol.
13:59-63[Medline].
|
| 34.
|
Singh, S.,
A. G. Sachs,
K. P. Chang, and S. G. Reed.
1995.
Diagnostic and prognostic value of k39 recombinant antigen in Indian leishmaniasis.
J. Parasitol.
81:1000-1003[CrossRef][Medline].
|
| 35.
|
Smyth, A. J.,
A. Ghosh,
Md. Q. Hassan,
D. Basu,
M. H. L. De Bruijn,
S. Adhya,
K. K. Mallik, and D. C. Barker.
1992.
Rapid and sensitive detection of Leishmania kinetoplast DNA from spleen and blood samples of kala-azar patients.
Parasitology
105:183-192.
|
| 36.
|
Sundar, S.,
S. G. Reed,
V. P. Singh,
P. C. K. Kumar, and H. W. Murray.
1998.
Rapid accurate field diagnosis of Indian visceral leishmaniasis.
Lancet
351:563-565[CrossRef][Medline].
|
| 37.
|
Thakur, C. P., and K. Kumar.
1992.
Post kala-azar dermal leishmaniasis: a neglected aspect of kala-azar control programmes.
Ann. Trop. Med. and Parasitol.
86:355-359.
|
| 38.
|
World Health Organization.
1990.
The leishmaniases. World Health Organization technical report series, no. 793.
World Health Organization, Geneva, Switzerland.
|
| 39.
|
Zijlstra, E. F.,
M. S. Ali,
A. M. El-Hassan,
I. A. El-Toum,
M. Satti,
H. W. Ghalib, and P. A. Kager.
1992.
Kala-azar; a comparative study of parasitological methods and the direct agglutination test in diagnosis.
Trans. R. Soc. Trop. Med. Hyg.
86:505-507[CrossRef][Medline].
|
Journal of Clinical Microbiology, March 2001, p. 849-854, Vol. 39, No. 3
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.3.849-854.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
