- Email: [email protected]
Molecular epidemiology of cutaneous leishmaniasis in Venezuela
TRANSACTIONSOFTHEROYALSOCIETYOFTROPICALMEDICINEANDHYGIENE(2002)96,SUPPLEMENT1S1/105-S1/109
Field epidemiology Molecular
epidemiology
of cutaneous
leishmaniasis
in Venezuela
Cruz M. Aguilati, Armando Rodriguez’, Douglas C. Barker3 and Noris Rodriguez’, Hector De Lima’, ‘Institute de Biomedicina, Universidad Central de Venezuela, Caracas, Venezuela,. ‘Universidad de Jacinto Convit’ Carabobo, Carabobo, Venezuela; 3Molteno Laboratories, Department OfPathology, University of Cambridge, Cambridge, UK Abstract This paper discusses the utility of a set of primers (3J1, 3J2) designed from a repetitive nuclear deoxvribonucleic acid seauence for the diagnosis of Leishmaniu braziliensis infection in samnles obtained from”humans, insect vectbrs and mammal& reservoir hosts from different endemic areas fn Venezuela. A high incidence of Leishmania (Viannia) braziliensis infection was found in the endemic areas studied. The sensitivity and specificity of the primers used were adequate for the identification of the natural vectors and reservoir hosts of L. (V.) bruziliensis. The polymerase chain reaction was more sensitive than culture and stained smear examination in the diagnosis of cutaneous leishmaniasis, detecting 80% of cases compared to 42% and 72%, respectively. Keywords: leishmaniasis, Leishmania braziliensis, epidemiology, diagnosis, Rattus ranus, Lutzomyia gomezi, Lutzomyia panamensis, Venezuela Introduction Leishmaniasis in Venezuela is a national health problem. The precise number of cases is not known, since 70% of the clinical cases are not confirmed after microscopical examination in the primary health service. Confirmatory parasitic diagnosis is needed, because cutaneous leishmaniasis co-exists with other tropical diseases with similar clinical manifestations and differential diagnosis is important in order to obtain rapid treatment and to understand fully the epidemiological situation and to design control measures. In Venezuela, Leishrnania braziliensis is considered the most important parasite from the epidemiological point of view. The parasite has been identified from human patients and domestic animals in several states (BONFANTE-GARRIDO etal., 1984,1991;G~w4~~1& TESH,~~~~;RODRIGUEZ etal., 1994). Despite the wide distribution of cutaneous leishmaniasis caused by L. bruziliensis”, the real epidemiological situation is unknown because of difficulties in identifying the parasite in humans, insect vectors and mammalian reservoirs. In the last few years, advances in molecular biology techniques have made possible the development of specific tools for diagnosis. The polymerase chain reaction (PCR) technique developed by MULLIS & FALOONA (1987) has been the most commonly used. This technique is based on the amplification in vitro of a specific fragment of deoxyribonucleic acid (DNA) by simultaneous extension of both chains of the DNA via the action of complementary primers and the enzyme DNA polymerase. Sev&ai authors have reported the use of the PCR in diaenosis of human leishmaniasis (ROGERS & WIRTH. ~~'?O;DEBRUIJN&BARKER,~~~~; ~~~~;RODRIGUE~ et al., 1994), using primers derived from kinetoplast DNA @DNA). Much effort has been made to develop species-specific DNA sequences for the specific diagnosis of the parasites producing different clinical manifestations of the disease. In this work we investigated the utility in field studies of a newly developed primer pair derived from nuclear DNA to evaluate the epidemiological situation of cuta-
Address for correspondence: Dr Noris Rodriguez, Instituto de Biomedicina, San Nicolas a Providencia, Apdo 4043, Caracas lOlOA, Venezuela; phone/fax +58 2 864 8624, e-mail [email protected] *Throughout this paper, the abbreviations L. and Lu. refer to Leishmania and Lutzonzyia, respectively.
neous leishmaniasis zuela.
in highly endemic
areas in Vene-
Materials and Methods Samples and DNA extraction For DNA extraction, biopsies taken in the field from skin lesions of human cases, samples of blood, liver and spleen of wild mammals, and pooled or individual sand flies were placed in Eppendorff tubes containing 100 & of TE buffer (10 mM Tris-HCl, pH 8 plus 10 mM ethylenediaminetetraacetic acid (EDTA)) and transported to the laboratory, where 5 yg/pL of proteinase K were added, the samples were heated at 56°C for 1 h, and then microfuged briefly. The supernatant was placed in a clean tube and an equal volume of phen&chloroform was added. After centrifugation for 10 min at 10 000 rev/min. the DNA was ureciuitated from the aqueous phise &h 100% ethanh and 4 mM LiCl at -2O”C, resuspended in TE buffer, and stored at 4°C until used. Giemsa-stained blood films and tissue impression smears were prepared and blood-agar cultures were also inoculated. DNA cloning and primer design A cloned fragment of 1.8 kb was isolated from an L. braziliensis total DNA genomic library (RODRIGUEZ et al., 1997) and sequenced using the ABITM apparatus (Applied Biosystems, model A373 A, version 1.2.1). After sequence analysis, 2 primers were designed: 3Jl (5’ TACCTGATGACTCCAC 3’, located between bp 751 and 800) and 3J2 (3’ CCTCATCA TACCGTTGATC 5’, the complimentary sequence, located downstream between bp 15 1 and 200). Two previously designed primers, B 1 and B2, which were specific for the L. bruziliensis complex and amplified a 700 bp fragment of kDNA, were also used. Polymerase chain reaction The PCR was carried out with 1 Ils, (100 ng) of total DNA in a reaction mixture containing 200 ng of each primer, 2.5 mM deoxynucleotides, and1 unit ?aq polymerase fGibco-BRL). The reaction mixture was submitted tb 35 amplification cvcles after 10 min at 95°C for total denaturation of the f>NA. Each cycle consisted of 1 min at 94°C for denaturation, 1 min at 60°C or 65°C (depending on the primers), -and 1 min at 72°C for extension. A final extension was performed for IO min. PCR products were analysed by gel electrophoresis in 1% agarose in TBE buffer (89 mM boric acid at
NORIS
S11106
pH 8.3, 90 mM Tris, 2.5 mM ethidium bromide (0.5 ug/uL), PolaroidTM 665 film.
EDTA), stained with and photographed on
DNA digestion and hybridization 10 pg of kDNA or total DNA were digested with 10 units of endonuclease (Gibco-BRL) according to the manufacturer’s instructions. The digested DNA fragments were separated by gel electrophoresis in 0.8% agarose. The separated fragments were blotted on to nitrocellulose membrane and ‘m-rally hybridized to an L. braziliensis species-specific probe (LbJ38) labelled with a32P-labelled d-adenosine triphosphate. Hybridization was carried out overnight, after which the filters were washed 3 times with 2X saline-sodium citrate buffer (SSC: 0.03 M Na citrate + 0.3 M Na Cl) and 05% sodium dodecyl sulphate at 65°C and exposed overnight at -80°C to blue-sensitive X-ray film (Molecular Technologies, St Louis, Missouri, USA). Table
1. Diagnosis
of Lekhmania
infection
Sigmodon hispidus Rattus rattus Didelphis marsupialis “No. positive/no. examined.
ETAL.
Results Examination of wild mammals The species and numbers of mammals trapped are shown in Table 1. The mammals were trapped during a one-week period each month and identified using conventional taxonomic criteria. Twenty-seven of 480 cultures were positive, and 4 were identified by kDNA restriction pattern and hybridization with species-specific probes as L. braziliensis (results not shown). The PCR results using primers 3Jl and 3J2 are shown in Fig. 1. The expected diagnostic band pattern was observed in 3 samples from R. rattus (lanes 1, 2 and 3) and in one sample from S. hispidus (lane 7). Two other samples from R. rattus did not amplify with these primers but gave positive results with primers specific for L. mexicana (results not shown). Sandfly collection and examination Sixty-five of 2700 dissected sand flies (Lutzomyia spp.) were naturally infected with flagellates in the midgut (Table 2).
in wild mammals
Mammals Species
RODRIGUEZ
in Venezuela Samples used for diagnosis
No. captured 391 tit
Fresh blood examination” 01107 15/30 (50%) 5118 (27.8%)
Stained blood film examination”
Blood culture examination”
01391 28168 (41.2%) 6131 (19.4%)
51391 (1.3%) 17/58 (29.3%) 5131 (16.1%)
k’n
1.35 1.07 0.87 0.60 0.50
Fig. 1. Polymerase chain reaction results using primers 3Jl and 3J2 with total DNA of parasites isolated from wild mammals in Venezuela and control strains. Lanes 1, 2 and 3, parasites from Rams rattus; lanes 4 and 7, parasites from Sigmodon hispidus; lane 5, Try~anosoma cmzi; lane 6, T. rangeli; lane 8, Leishmania mexicana (Be1 21); lane 9, L. amazonensis (PHS); lane 10, L. enriettii (L88); lane 11, L. guyanensis (M4147); lane 12, L. bradiensis (LTB300); lane 13, negative control; lane M, molecular size markers.
CUTANEOUS
LEISHMANIASIS
IN VENEZUELA
Table 2. Numbers of Lutzomyia mastigotes in the midgut
s11107
spp. with
pro-
Species
No. infected
Lu. cayennensis Lu. trinidadensis Lu. panamensis Lu. gomezi
51 (78.5%)
7 5 2 65
Total” “2700
were negative with primers 3Jl and 3J2 (Fig. 3, B). Further kDNA restriction pattern analysis and DNA hvbridization showed this uarasite to be L. mvanensis. esing the kDNA primers 81 and B2, 74 of ~dsamples gave positive results, as did 70 of 90 when the speciesspecific primers 3Jl were used. The identity of the human isolates was confirmed as L. (V.) braziliensis bv hybridization of the digested genomic’ DNA (Fig. 4). Overall. the PCR method usine mimers 311 and 312 was 8% more sensitive than ex>mination “of stained smears and 42% more sensitive than cultivation in vitro for the diagnosis of cutaneous leishmaniasis (Fig. 5).
(108%) (7.7%) (3.1%) (100%)
sand flies were examined.
Discussion Members of the L. braziliensis complex are me causative agents of localized human cutaneous leishmaniasis. Accurate identification of the species of Leishmania is needed for a variety of clinical and epidemiological reasons, and DNA-based techniques have recently been investigated as potential tools for this purpose (BARKER, 1989; VAN EYS et al., 1992). Preliminary data based on minicircle DNA restriction pattern and Southern blotting analysis have demonstrated that the patterns of isolates belonging to the same taxa have some similarities (JAcKsoN~~~Z., 1984; SPITHILL & GRUMOT, 1984). Analvsis of kDNA seems to emphasize dissimilarities rather man relationships between strains. Identification of Leishmania species should be based on DNA sequences that have a high degree of homology within a taxon and polymorphism of restriction patterns between taxa. In this
The results obtained after 35 cycles of amplification using primers 3Jl and 3J2 with total DNA isolated from individual and pooled sand flies are shown in Fig. 2. The expected diagnostic band (506 bp) was observed in samples from Lu. panamensis and Lu. gomezi only. Human
leishmaniasis
The sensitivity of the PCR technique was compared with conventional methods. Amastigotes were identitied in 65 of 90 stained biopsy preparations and promastigotes developed in 38 of 90 biopsy cultures. The PCR was carried out with primers Bl and B2 (specific for the L. braziliensis complex) and with primers 3Jl and 3J2 (specific for L. (IX) braziliensis) (Fig. 3). The sample in lanes 1 and 6 of Fig. 3 gave positive results with primers El and B2, but the same samples 1
23
45
67
S
9
10
I1
12
13
14
15
16
I7
18
M
bp
Fig. 2. Polymerase chain reaction products obtained after 35 amplification cycles with total DNA of Lutzomyia spp. and control strains of Leishmania and Tiy~~~nosoma. Products were separated in 1% agarose. Lanes 1, 7, 12 and 16, Lu. gomezi; lane 2, Lu. gomezi (pool); lanes 3, 8 and 13, Lu. cuyennensis; lane 4, Lu. panamensis; lanes 5, 10 and 11, Lu. trinidadensis; lane 6, Lu. cayennensis (pool); lane 14, T. cruzi; lane 15, T. rangeli; lane 17, L. braziliensis LTB300 (positive control); lane 18, negative control; lane M, molecular size markers.
NORIS RODRIGUEZ
S1/108
ETAL.
bp
1353 1078 -_I 872 603
- 7i 30
506bp
31G
Fig. 3. Polymerase chain reaction results using primers 3Jl and 352 (panel A) and Bl and B2 (panel B) with DNA samples from patients with leishmaniasis. Lanes l- 11, total DNA from biopsies of skin lesions; lane 12, Leishrnaniu braziliensis LTBSOO (positive control); lane 13, negative control; lane M, molecular size markers. The samples in lanes 1 and 6 were later shown to be L. guyanensis.
1
2
3
4
5
1
2
3
4
5
kb
- 2.03
B
,
Fig. 4. Total DNA samples, digested with restriction endonuclease PstI and separated on 0.8% agarose, from patients with cutaneous leishmaniasis (lanes l-3), L. (V.) bruziliensis LTBSOO (lane 4) and L. (V.) guyanensis M4147 (lane 5). Panel A: stained with ethidium bromide (0.5% l.tg/mL); panel B after overnight hybridization with probe LbJ38 and overnight exposure to X-ray film.
Diagnostic
work we used different molecular approaches to identify Leishrnaniu in samples obtained from humans, sand flies and wild mammals. Our results have shown the usefulness of the PCR hybridization techniques in elucidating: the enidemiolonical situation in different areas. The’ note&al of the-techniques to identify the parasite in individual and pooled sand fly samples indicates that these methods, using species-specific probes, can be used to identify the natural vectors of leishmaniasis. This has previously been difficult, because of difficulties in identifying- the parasites within the sand flies due to their mornholoeical similaritv and also to the use of primers de&ed fFom the conserved region of kDNA, which may not differentiate between different members of the subgenus L. (Vknrziu). BARRIOS et al. (1994) have demonstrated the coexistence of both subgenera of Leishmaniu (T&m& and Leishmania) in the same sand fly, further emphasizing the need for accurate specific identification in the vector. Our results have suggested that Lu. gomezi and Lu. panamensis are possible vectors of cutaneous leishmaniasis in the Carabobo endemic area, as previously demonstrated bv RODRIGUEZ et al. (1999). We have shown earlier that Lu. gomezi is the vector of L. braziliensis in an endemic area of Miranda State, Venezuela, using kDNA orimers and hvbridization techniaues (FELICIANGE~I et al., 1994).-The results here rep’orted are a further demonstration of the role of this species in the transmission of L. braziliensis in Venezuelan endemic areas. The PCR technique has also allowed the identification of natural reservoirs of L. braziliensis, R. rattus and S. hispidus, in Lara State, Venezuela. These results were confirmed after hybridization and study of kDNA restriction patterns obtained after culturing of the parasites isolated from the same samples (results not shown). Other epidemiological studies in Merida State (paper in preparation) have confirmed the role of R. rattus as a reservoir of L. braziliensis. The sensitivity of the PCR-based test for detection of human cutaneous leishmaniasis was assessed in this work using biopsies from human patients with skin lesions and clinical and immunological indications of
Test
Fig. 5. Efficiency of different diagnostic procedures for Leishmania braziliensis infection: stained blood film (‘smear’), blood culture and polymerase chain reaction (PCR) using nuclear and kinetoplast DNA primers (3J1/3J2 and BlIB2, respectively) .
cutaneous
leishmaniasis.
The
results
I
obtained
with
the
genomic DNA primers 3Jl and 3J2 and the kDNA primers Bl and B2 were generally concordant with microscopical observation of amastigotes in tissue smears (the ‘gold standard’ diagnostic method), which was positive in 72% of the samples from human biopsies. In our hands the 2 PCR methods gave positive
CUTANEOUS
LEISHMANJASIS
IN VENEZUELA
I
results in 80-84% of the samples. The PCR methbd using primers based on kDNA was 4% more sensitibe than that using genomic DNA assay primers. Howev$r, the nenomic DNA nrimers (311 and 3T2) were more spe&c at the lowei taxono& level, permitting he specific identification of L. (V.) braziliensis without the need to use hybridization for the parasite. Our results are in DE BRUIJN et al. (1993), who reported that the P technique was more sensitive nostic methods, giving positive results with samples. The results reported in this paper constitute the fi st demonstration of the comnlete enidemiolom of IL. braziliensis in Venezuela, anh were- achieved-&th the aid of molecular techniques. The use of these techqiques simplifies epidemiological studies in which it :is necessary to process large number of samples to find’ a single individual, vector or reservoir host, natura i ly infected with Leishmania. The DNA-derived primers 3Jl and 3J2 are speciesspecific and we have shown them to be very useful i.n epidemiological studies of cutaneous 1eishmanias”s. Using both sets of primers we have elucidated t ;be epidemiology of L. braziliensis in different endembc areas in Venezuela. However. mimers 3Tl and 372 aie more sensitive and specific fdr&L. brazili&sis in v&io&s biological samples, and have the advantage of savidg time in the identification of natural vectors and resekvoirs of this parasite, whereas the conventional techniques are less successful because they are non-specifik, tedius and time-consuming. Acknowledgements This work received projects Ven/97/002/016
financial support from andVeni971002/02124.
World
BaIpk
References Barker, D. C. (1989). Molecular approachs to DNA diagnosis. Parasitology, 99, 125-146. Barrios, M. A., Rodriguez, N., Feliciangeli, M. D., Ulricb, M., Telles, S., Pinardi, M. E. & Convit, J. (1994). Coexistence of two species of Leishmania in the digestive tract of the vector Lutzonzyia ovallesi. American Journal of Tropicpl Medicine and Hygiene, 51, 669-675. Bonfante-Garrido, R., Mejia De Alejos, M. A., Melendez, I$, Arredondo, C. & Urdaneta, R. (1984). Tegumentary urban leishmaniasis in Barquisimeto, Venezuela. PAHO Bulletin, 97,105110.
s11109
Bonfante-Garrido, R., Urdaneta, I., Urdaneta, R. & Alvarado, 1. 11991). Natural infection of cats with Leishmania in bar&isi&eto, Venezuela. Transactions of the Royal Society of Tropical Medicine and Hygiene, 85, 53. De Bruijn, M. H. L. & Barker, D. C. (1992). Diagnosis of New World leishmaniasis: specific detection of the Leishmania bruziliensis complex by amplification of kinetoplast DNA. Acta Tropica, 52,45-58. De Bruijn, M. H. L., Labrada, L. A., Smyth, A. J. & Barker, D. C. (1993). A comparative study of diagnosis by the polymerase chain reaction and by current clinical methods using biopsies from Colombian patients with suspected leishmaniasis. Tropical Medicine and Parasitology, 44, 201-201. Feliciangeli, M. D., Rodriguez, N., Bravo, A, Arias, F. & Guzman, B. (1994). Vectors of cutaneous lelshmaniasis in north-central Venezuela. Medical and Veterinary Entomology, 8,317-324. Grimaldi, G., jr & Tesh, R. B. (1993). Leishmania of the New World: current concepts and implications for future research. Clinical Microbiology Reviews, 83, 230-250. Jackson, I’., Wohlhieter, J., Jackson, J., Sayles, I’., Diggs, C. & Hockmeyer, W. (1984). Restriction endonuclease analysis of Leishmania kinetoplast DNA characterizes parasites responsible for visceral and cutaneous disease. American Journal of Tropical Medicine and Hygiene, 33, 808-819. Mullis. I<. B. & Faloona. F. A. (19871. Snecific svnthesis of DNk in vitro via a p&ymera&-caialyzkd chaii reaction. Methods in Enzymology, 155, 335-360. Rodriguez, N., Guzman, B., Rodas, A., Takiff H., Bloom, B. & Convit, J. (1994). Diagnosis of cutaneous leishmamasis and suecies discrimination of oarasite bv PCR and hvbridization. Journal of Clinical Microbiology, 9; 2246-2252.Rodriguez, N., De Lima, H., Rodriguez, A., Brewster, S. & Barker, D. C. (1997). Identification of a repetitive DNA sequence from Leishmania (Viannia) braziliensis, Venezuelan strain. Parasitology, 115, 349-358. Rodriguez, N., Aguilar, C. M., Barrios, M. A. & Barker, D. C. (1999). Identification of Leishmania braziliensis in naturally infected individual sandflies. Transactions of the Royal Society of Tropical Medicine and Hygiene, 93,47-49. Rogers, M. S. & Wirth, D. F. (1990). Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania. Experimental Parasitology, 71,267-275. Spithill, T. W. & Grumot, R. J. (1984). Identification of species, strains and clones of Leishmania by characterization of kinetoplast DNA minicircles. Molecular and Biochemical Parasitology, 12, 2 17-223. Van Eys, G. J. J. M., Schoone, G. J., Kroon, N. C. M. & Ebeling, S. B. (1992). Sequence analysis of small sub-unit ribosomal RNA genes and its use for detection and identification of Leishmania parasites. Molecular and Biochemical Parasitology, 51, 133-142.
Field epidemiology Molecular
epidemiology
of cutaneous
leishmaniasis
in Venezuela
Cruz M. Aguilati, Armando Rodriguez’, Douglas C. Barker3 and Noris Rodriguez’, Hector De Lima’, ‘Institute de Biomedicina, Universidad Central de Venezuela, Caracas, Venezuela,. ‘Universidad de Jacinto Convit’ Carabobo, Carabobo, Venezuela; 3Molteno Laboratories, Department OfPathology, University of Cambridge, Cambridge, UK Abstract This paper discusses the utility of a set of primers (3J1, 3J2) designed from a repetitive nuclear deoxvribonucleic acid seauence for the diagnosis of Leishmaniu braziliensis infection in samnles obtained from”humans, insect vectbrs and mammal& reservoir hosts from different endemic areas fn Venezuela. A high incidence of Leishmania (Viannia) braziliensis infection was found in the endemic areas studied. The sensitivity and specificity of the primers used were adequate for the identification of the natural vectors and reservoir hosts of L. (V.) bruziliensis. The polymerase chain reaction was more sensitive than culture and stained smear examination in the diagnosis of cutaneous leishmaniasis, detecting 80% of cases compared to 42% and 72%, respectively. Keywords: leishmaniasis, Leishmania braziliensis, epidemiology, diagnosis, Rattus ranus, Lutzomyia gomezi, Lutzomyia panamensis, Venezuela Introduction Leishmaniasis in Venezuela is a national health problem. The precise number of cases is not known, since 70% of the clinical cases are not confirmed after microscopical examination in the primary health service. Confirmatory parasitic diagnosis is needed, because cutaneous leishmaniasis co-exists with other tropical diseases with similar clinical manifestations and differential diagnosis is important in order to obtain rapid treatment and to understand fully the epidemiological situation and to design control measures. In Venezuela, Leishrnania braziliensis is considered the most important parasite from the epidemiological point of view. The parasite has been identified from human patients and domestic animals in several states (BONFANTE-GARRIDO etal., 1984,1991;G~w4~~1& TESH,~~~~;RODRIGUEZ etal., 1994). Despite the wide distribution of cutaneous leishmaniasis caused by L. bruziliensis”, the real epidemiological situation is unknown because of difficulties in identifying the parasite in humans, insect vectors and mammalian reservoirs. In the last few years, advances in molecular biology techniques have made possible the development of specific tools for diagnosis. The polymerase chain reaction (PCR) technique developed by MULLIS & FALOONA (1987) has been the most commonly used. This technique is based on the amplification in vitro of a specific fragment of deoxyribonucleic acid (DNA) by simultaneous extension of both chains of the DNA via the action of complementary primers and the enzyme DNA polymerase. Sev&ai authors have reported the use of the PCR in diaenosis of human leishmaniasis (ROGERS & WIRTH. ~~'?O;DEBRUIJN&BARKER,~~~~; ~~~~;RODRIGUE~ et al., 1994), using primers derived from kinetoplast DNA @DNA). Much effort has been made to develop species-specific DNA sequences for the specific diagnosis of the parasites producing different clinical manifestations of the disease. In this work we investigated the utility in field studies of a newly developed primer pair derived from nuclear DNA to evaluate the epidemiological situation of cuta-
Address for correspondence: Dr Noris Rodriguez, Instituto de Biomedicina, San Nicolas a Providencia, Apdo 4043, Caracas lOlOA, Venezuela; phone/fax +58 2 864 8624, e-mail [email protected] *Throughout this paper, the abbreviations L. and Lu. refer to Leishmania and Lutzonzyia, respectively.
neous leishmaniasis zuela.
in highly endemic
areas in Vene-
Materials and Methods Samples and DNA extraction For DNA extraction, biopsies taken in the field from skin lesions of human cases, samples of blood, liver and spleen of wild mammals, and pooled or individual sand flies were placed in Eppendorff tubes containing 100 & of TE buffer (10 mM Tris-HCl, pH 8 plus 10 mM ethylenediaminetetraacetic acid (EDTA)) and transported to the laboratory, where 5 yg/pL of proteinase K were added, the samples were heated at 56°C for 1 h, and then microfuged briefly. The supernatant was placed in a clean tube and an equal volume of phen&chloroform was added. After centrifugation for 10 min at 10 000 rev/min. the DNA was ureciuitated from the aqueous phise &h 100% ethanh and 4 mM LiCl at -2O”C, resuspended in TE buffer, and stored at 4°C until used. Giemsa-stained blood films and tissue impression smears were prepared and blood-agar cultures were also inoculated. DNA cloning and primer design A cloned fragment of 1.8 kb was isolated from an L. braziliensis total DNA genomic library (RODRIGUEZ et al., 1997) and sequenced using the ABITM apparatus (Applied Biosystems, model A373 A, version 1.2.1). After sequence analysis, 2 primers were designed: 3Jl (5’ TACCTGATGACTCCAC 3’, located between bp 751 and 800) and 3J2 (3’ CCTCATCA TACCGTTGATC 5’, the complimentary sequence, located downstream between bp 15 1 and 200). Two previously designed primers, B 1 and B2, which were specific for the L. bruziliensis complex and amplified a 700 bp fragment of kDNA, were also used. Polymerase chain reaction The PCR was carried out with 1 Ils, (100 ng) of total DNA in a reaction mixture containing 200 ng of each primer, 2.5 mM deoxynucleotides, and1 unit ?aq polymerase fGibco-BRL). The reaction mixture was submitted tb 35 amplification cvcles after 10 min at 95°C for total denaturation of the f>NA. Each cycle consisted of 1 min at 94°C for denaturation, 1 min at 60°C or 65°C (depending on the primers), -and 1 min at 72°C for extension. A final extension was performed for IO min. PCR products were analysed by gel electrophoresis in 1% agarose in TBE buffer (89 mM boric acid at
NORIS
S11106
pH 8.3, 90 mM Tris, 2.5 mM ethidium bromide (0.5 ug/uL), PolaroidTM 665 film.
EDTA), stained with and photographed on
DNA digestion and hybridization 10 pg of kDNA or total DNA were digested with 10 units of endonuclease (Gibco-BRL) according to the manufacturer’s instructions. The digested DNA fragments were separated by gel electrophoresis in 0.8% agarose. The separated fragments were blotted on to nitrocellulose membrane and ‘m-rally hybridized to an L. braziliensis species-specific probe (LbJ38) labelled with a32P-labelled d-adenosine triphosphate. Hybridization was carried out overnight, after which the filters were washed 3 times with 2X saline-sodium citrate buffer (SSC: 0.03 M Na citrate + 0.3 M Na Cl) and 05% sodium dodecyl sulphate at 65°C and exposed overnight at -80°C to blue-sensitive X-ray film (Molecular Technologies, St Louis, Missouri, USA). Table
1. Diagnosis
of Lekhmania
infection
Sigmodon hispidus Rattus rattus Didelphis marsupialis “No. positive/no. examined.
ETAL.
Results Examination of wild mammals The species and numbers of mammals trapped are shown in Table 1. The mammals were trapped during a one-week period each month and identified using conventional taxonomic criteria. Twenty-seven of 480 cultures were positive, and 4 were identified by kDNA restriction pattern and hybridization with species-specific probes as L. braziliensis (results not shown). The PCR results using primers 3Jl and 3J2 are shown in Fig. 1. The expected diagnostic band pattern was observed in 3 samples from R. rattus (lanes 1, 2 and 3) and in one sample from S. hispidus (lane 7). Two other samples from R. rattus did not amplify with these primers but gave positive results with primers specific for L. mexicana (results not shown). Sandfly collection and examination Sixty-five of 2700 dissected sand flies (Lutzomyia spp.) were naturally infected with flagellates in the midgut (Table 2).
in wild mammals
Mammals Species
RODRIGUEZ
in Venezuela Samples used for diagnosis
No. captured 391 tit
Fresh blood examination” 01107 15/30 (50%) 5118 (27.8%)
Stained blood film examination”
Blood culture examination”
01391 28168 (41.2%) 6131 (19.4%)
51391 (1.3%) 17/58 (29.3%) 5131 (16.1%)
k’n
1.35 1.07 0.87 0.60 0.50
Fig. 1. Polymerase chain reaction results using primers 3Jl and 3J2 with total DNA of parasites isolated from wild mammals in Venezuela and control strains. Lanes 1, 2 and 3, parasites from Rams rattus; lanes 4 and 7, parasites from Sigmodon hispidus; lane 5, Try~anosoma cmzi; lane 6, T. rangeli; lane 8, Leishmania mexicana (Be1 21); lane 9, L. amazonensis (PHS); lane 10, L. enriettii (L88); lane 11, L. guyanensis (M4147); lane 12, L. bradiensis (LTB300); lane 13, negative control; lane M, molecular size markers.
CUTANEOUS
LEISHMANIASIS
IN VENEZUELA
Table 2. Numbers of Lutzomyia mastigotes in the midgut
s11107
spp. with
pro-
Species
No. infected
Lu. cayennensis Lu. trinidadensis Lu. panamensis Lu. gomezi
51 (78.5%)
7 5 2 65
Total” “2700
were negative with primers 3Jl and 3J2 (Fig. 3, B). Further kDNA restriction pattern analysis and DNA hvbridization showed this uarasite to be L. mvanensis. esing the kDNA primers 81 and B2, 74 of ~dsamples gave positive results, as did 70 of 90 when the speciesspecific primers 3Jl were used. The identity of the human isolates was confirmed as L. (V.) braziliensis bv hybridization of the digested genomic’ DNA (Fig. 4). Overall. the PCR method usine mimers 311 and 312 was 8% more sensitive than ex>mination “of stained smears and 42% more sensitive than cultivation in vitro for the diagnosis of cutaneous leishmaniasis (Fig. 5).
(108%) (7.7%) (3.1%) (100%)
sand flies were examined.
Discussion Members of the L. braziliensis complex are me causative agents of localized human cutaneous leishmaniasis. Accurate identification of the species of Leishmania is needed for a variety of clinical and epidemiological reasons, and DNA-based techniques have recently been investigated as potential tools for this purpose (BARKER, 1989; VAN EYS et al., 1992). Preliminary data based on minicircle DNA restriction pattern and Southern blotting analysis have demonstrated that the patterns of isolates belonging to the same taxa have some similarities (JAcKsoN~~~Z., 1984; SPITHILL & GRUMOT, 1984). Analvsis of kDNA seems to emphasize dissimilarities rather man relationships between strains. Identification of Leishmania species should be based on DNA sequences that have a high degree of homology within a taxon and polymorphism of restriction patterns between taxa. In this
The results obtained after 35 cycles of amplification using primers 3Jl and 3J2 with total DNA isolated from individual and pooled sand flies are shown in Fig. 2. The expected diagnostic band (506 bp) was observed in samples from Lu. panamensis and Lu. gomezi only. Human
leishmaniasis
The sensitivity of the PCR technique was compared with conventional methods. Amastigotes were identitied in 65 of 90 stained biopsy preparations and promastigotes developed in 38 of 90 biopsy cultures. The PCR was carried out with primers Bl and B2 (specific for the L. braziliensis complex) and with primers 3Jl and 3J2 (specific for L. (IX) braziliensis) (Fig. 3). The sample in lanes 1 and 6 of Fig. 3 gave positive results with primers El and B2, but the same samples 1
23
45
67
S
9
10
I1
12
13
14
15
16
I7
18
M
bp
Fig. 2. Polymerase chain reaction products obtained after 35 amplification cycles with total DNA of Lutzomyia spp. and control strains of Leishmania and Tiy~~~nosoma. Products were separated in 1% agarose. Lanes 1, 7, 12 and 16, Lu. gomezi; lane 2, Lu. gomezi (pool); lanes 3, 8 and 13, Lu. cuyennensis; lane 4, Lu. panamensis; lanes 5, 10 and 11, Lu. trinidadensis; lane 6, Lu. cayennensis (pool); lane 14, T. cruzi; lane 15, T. rangeli; lane 17, L. braziliensis LTB300 (positive control); lane 18, negative control; lane M, molecular size markers.
NORIS RODRIGUEZ
S1/108
ETAL.
bp
1353 1078 -_I 872 603
- 7i 30
506bp
31G
Fig. 3. Polymerase chain reaction results using primers 3Jl and 352 (panel A) and Bl and B2 (panel B) with DNA samples from patients with leishmaniasis. Lanes l- 11, total DNA from biopsies of skin lesions; lane 12, Leishrnaniu braziliensis LTBSOO (positive control); lane 13, negative control; lane M, molecular size markers. The samples in lanes 1 and 6 were later shown to be L. guyanensis.
1
2
3
4
5
1
2
3
4
5
kb
- 2.03
B
,
Fig. 4. Total DNA samples, digested with restriction endonuclease PstI and separated on 0.8% agarose, from patients with cutaneous leishmaniasis (lanes l-3), L. (V.) bruziliensis LTBSOO (lane 4) and L. (V.) guyanensis M4147 (lane 5). Panel A: stained with ethidium bromide (0.5% l.tg/mL); panel B after overnight hybridization with probe LbJ38 and overnight exposure to X-ray film.
Diagnostic
work we used different molecular approaches to identify Leishrnaniu in samples obtained from humans, sand flies and wild mammals. Our results have shown the usefulness of the PCR hybridization techniques in elucidating: the enidemiolonical situation in different areas. The’ note&al of the-techniques to identify the parasite in individual and pooled sand fly samples indicates that these methods, using species-specific probes, can be used to identify the natural vectors of leishmaniasis. This has previously been difficult, because of difficulties in identifying- the parasites within the sand flies due to their mornholoeical similaritv and also to the use of primers de&ed fFom the conserved region of kDNA, which may not differentiate between different members of the subgenus L. (Vknrziu). BARRIOS et al. (1994) have demonstrated the coexistence of both subgenera of Leishmaniu (T&m& and Leishmania) in the same sand fly, further emphasizing the need for accurate specific identification in the vector. Our results have suggested that Lu. gomezi and Lu. panamensis are possible vectors of cutaneous leishmaniasis in the Carabobo endemic area, as previously demonstrated bv RODRIGUEZ et al. (1999). We have shown earlier that Lu. gomezi is the vector of L. braziliensis in an endemic area of Miranda State, Venezuela, using kDNA orimers and hvbridization techniaues (FELICIANGE~I et al., 1994).-The results here rep’orted are a further demonstration of the role of this species in the transmission of L. braziliensis in Venezuelan endemic areas. The PCR technique has also allowed the identification of natural reservoirs of L. braziliensis, R. rattus and S. hispidus, in Lara State, Venezuela. These results were confirmed after hybridization and study of kDNA restriction patterns obtained after culturing of the parasites isolated from the same samples (results not shown). Other epidemiological studies in Merida State (paper in preparation) have confirmed the role of R. rattus as a reservoir of L. braziliensis. The sensitivity of the PCR-based test for detection of human cutaneous leishmaniasis was assessed in this work using biopsies from human patients with skin lesions and clinical and immunological indications of
Test
Fig. 5. Efficiency of different diagnostic procedures for Leishmania braziliensis infection: stained blood film (‘smear’), blood culture and polymerase chain reaction (PCR) using nuclear and kinetoplast DNA primers (3J1/3J2 and BlIB2, respectively) .
cutaneous
leishmaniasis.
The
results
I
obtained
with
the
genomic DNA primers 3Jl and 3J2 and the kDNA primers Bl and B2 were generally concordant with microscopical observation of amastigotes in tissue smears (the ‘gold standard’ diagnostic method), which was positive in 72% of the samples from human biopsies. In our hands the 2 PCR methods gave positive
CUTANEOUS
LEISHMANJASIS
IN VENEZUELA
I
results in 80-84% of the samples. The PCR methbd using primers based on kDNA was 4% more sensitibe than that using genomic DNA assay primers. Howev$r, the nenomic DNA nrimers (311 and 3T2) were more spe&c at the lowei taxono& level, permitting he specific identification of L. (V.) braziliensis without the need to use hybridization for the parasite. Our results are in DE BRUIJN et al. (1993), who reported that the P technique was more sensitive nostic methods, giving positive results with samples. The results reported in this paper constitute the fi st demonstration of the comnlete enidemiolom of IL. braziliensis in Venezuela, anh were- achieved-&th the aid of molecular techniques. The use of these techqiques simplifies epidemiological studies in which it :is necessary to process large number of samples to find’ a single individual, vector or reservoir host, natura i ly infected with Leishmania. The DNA-derived primers 3Jl and 3J2 are speciesspecific and we have shown them to be very useful i.n epidemiological studies of cutaneous 1eishmanias”s. Using both sets of primers we have elucidated t ;be epidemiology of L. braziliensis in different endembc areas in Venezuela. However. mimers 3Tl and 372 aie more sensitive and specific fdr&L. brazili&sis in v&io&s biological samples, and have the advantage of savidg time in the identification of natural vectors and resekvoirs of this parasite, whereas the conventional techniques are less successful because they are non-specifik, tedius and time-consuming. Acknowledgements This work received projects Ven/97/002/016
financial support from andVeni971002/02124.
World
BaIpk
References Barker, D. C. (1989). Molecular approachs to DNA diagnosis. Parasitology, 99, 125-146. Barrios, M. A., Rodriguez, N., Feliciangeli, M. D., Ulricb, M., Telles, S., Pinardi, M. E. & Convit, J. (1994). Coexistence of two species of Leishmania in the digestive tract of the vector Lutzonzyia ovallesi. American Journal of Tropicpl Medicine and Hygiene, 51, 669-675. Bonfante-Garrido, R., Mejia De Alejos, M. A., Melendez, I$, Arredondo, C. & Urdaneta, R. (1984). Tegumentary urban leishmaniasis in Barquisimeto, Venezuela. PAHO Bulletin, 97,105110.
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Bonfante-Garrido, R., Urdaneta, I., Urdaneta, R. & Alvarado, 1. 11991). Natural infection of cats with Leishmania in bar&isi&eto, Venezuela. Transactions of the Royal Society of Tropical Medicine and Hygiene, 85, 53. De Bruijn, M. H. L. & Barker, D. C. (1992). Diagnosis of New World leishmaniasis: specific detection of the Leishmania bruziliensis complex by amplification of kinetoplast DNA. Acta Tropica, 52,45-58. De Bruijn, M. H. L., Labrada, L. A., Smyth, A. J. & Barker, D. C. (1993). A comparative study of diagnosis by the polymerase chain reaction and by current clinical methods using biopsies from Colombian patients with suspected leishmaniasis. Tropical Medicine and Parasitology, 44, 201-201. Feliciangeli, M. D., Rodriguez, N., Bravo, A, Arias, F. & Guzman, B. (1994). Vectors of cutaneous lelshmaniasis in north-central Venezuela. Medical and Veterinary Entomology, 8,317-324. Grimaldi, G., jr & Tesh, R. B. (1993). Leishmania of the New World: current concepts and implications for future research. Clinical Microbiology Reviews, 83, 230-250. Jackson, I’., Wohlhieter, J., Jackson, J., Sayles, I’., Diggs, C. & Hockmeyer, W. (1984). Restriction endonuclease analysis of Leishmania kinetoplast DNA characterizes parasites responsible for visceral and cutaneous disease. American Journal of Tropical Medicine and Hygiene, 33, 808-819. Mullis. I<. B. & Faloona. F. A. (19871. Snecific svnthesis of DNk in vitro via a p&ymera&-caialyzkd chaii reaction. Methods in Enzymology, 155, 335-360. Rodriguez, N., Guzman, B., Rodas, A., Takiff H., Bloom, B. & Convit, J. (1994). Diagnosis of cutaneous leishmamasis and suecies discrimination of oarasite bv PCR and hvbridization. Journal of Clinical Microbiology, 9; 2246-2252.Rodriguez, N., De Lima, H., Rodriguez, A., Brewster, S. & Barker, D. C. (1997). Identification of a repetitive DNA sequence from Leishmania (Viannia) braziliensis, Venezuelan strain. Parasitology, 115, 349-358. Rodriguez, N., Aguilar, C. M., Barrios, M. A. & Barker, D. C. (1999). Identification of Leishmania braziliensis in naturally infected individual sandflies. Transactions of the Royal Society of Tropical Medicine and Hygiene, 93,47-49. Rogers, M. S. & Wirth, D. F. (1990). Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania. Experimental Parasitology, 71,267-275. Spithill, T. W. & Grumot, R. J. (1984). Identification of species, strains and clones of Leishmania by characterization of kinetoplast DNA minicircles. Molecular and Biochemical Parasitology, 12, 2 17-223. Van Eys, G. J. J. M., Schoone, G. J., Kroon, N. C. M. & Ebeling, S. B. (1992). Sequence analysis of small sub-unit ribosomal RNA genes and its use for detection and identification of Leishmania parasites. Molecular and Biochemical Parasitology, 51, 133-142.