Acta Tropica 100 (2006) 241–245

Short communication

Species-specific PCR assay for L. infantum/L. donovani

discrimination夽
Mallorie Hide ∗ , Anne-Laure Bañuls
IRD de Montpellier, Laboratory GEMI, UMR CNRS/IRD 2724, 911, avenue Agropolis BP 64501, 34394 Montpellier Cedex 5, France
Received 20 December 2005; received in revised form 25 October 2006; accepted 26 October 2006
Available online 4 December 2006

Abstract
Leishmania infantum and Leishmania donovani both pertain to the L. (L.) donovani complex. The status of certain strains is
questioned in the literature and there are no reliable discriminative markers to identify them. Molecular tools are needed to (i)
identify diagnostic markers and (ii) to allow a better understanding of phylogenetic relationships. We have developed a PCR based
on cysteine protease B (cpb). This PCR discriminates between L. infantum and L. donovani with 50–100 pg of DNA. These two
species are differentiated by their fragment length. Indeed, L. donovani strains were characterized by a 741-bp product and L.
infantum strains by a 702-bp product, except for one strain, which revealed a heterozygous profile with the two products. This PCR
does not generate amplification for other Leishmania or kinetoplastids and could contribute to clarify the phylogenetic status of
several taxa that are also being debated, such as L. archibaldi.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Cysteine protease B; L. donovani complex; Polymerase chain reaction

1. Introduction species of the Leishmania donovani complex. This

visceral form is characterized by hepatosplenomegaly,
Leishmania is a trypanosomatid responsible for leish- anemia, immunosuppression, hypergammaglobulinemia
maniasis. This disease ranges in severity from a healing and fever. The reference standard technique for Leishma-
skin ulcer to an overwhelming visceral form. Leishma- nia species typing is multilocus enzyme electrophoresis
niasis is a worldwide endemic disease, with an estimated (MLEE) (Hide et al., 2001; Rioux et al., 1990). However,
disease burden of 2,357,000 disability-adjusted life years this method is not ideal, as it does not easily differenti-
and 59,000 deaths per year (WHO, 2002). The most ate species within the L. donovani complex and requires
severe form (visceral leishmaniasis) is caused by the a large amount of parasite. Indeed, the only isoen-
zymatic system (glutamate oxaloacetate transaminase,
GOT E.C.2.6.1.1) that was supposed to give a discrim-
夽 Note: Nucleotide sequence data reported in this paper are available inative pattern within the complex, has been shown not
in the GenBank database under accession numbers AY896776 to be reliable, because some Sudanese strains identified
AY896777, AY896778, AY896779, AY896780, AY896781, as Leishmania infantum by GOT locus were shown to
AY896782, AY896783, AY896784, AY896785, AY896786, actually be L. donovani strains (Jamjoom et al., 2004).
AY896787, AY896788, AY896789, AY896790, AY896791.
∗ Corresponding author. Tel.: +33 4 67 41 61 80; It is thus necessary to supplement this classical MLEE
fax: +33 4 67 41 62 99. typing by specific PCR. The discrimination between L.
E-mail address: [email protected] (M. Hide). infantum and L. donovani is important because its taxa

0001-706X/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.actatropica.2006.10.012
242 M. Hide, A.-L. Bañuls / Acta Tropica 100 (2006) 241–245

are morphologically indistinguishable but are associ- 2.4. Specific PCR of cpbEF copies
ated with different epidemiology, ecology and pathology.
L. infantum is anthropozoonotic with a dog reservoir, Genomic DNA was extracted from parasite pellets
whereas L. donovani is largely anthroponotic. To develop and clinical samples by classical phenol/chloroform
a discriminative PCR, we focused on the cathepsin-l extraction and from parasite culture (1 ml) by
proteases CPB belonging to the papain-like superfamily, InstaGeneTM Matrix (Bio-Rad). For the second method,
clan CA and family C1. They have attracted considerable after centrifugation, 200 ␮l of InstaGeneTM matrix was
attention because of their role in destruction of host pro- added to the pellet and incubated 15 min at 56 ◦ C, then
tein and evasion of host immune response (Alexander heated 8 min at 100 ◦ C.
et al., 1998). CPB enzymes are encoded by a tandem The optimal conditions for cpbEF amplifica-
array located in a single locus. Within the L. donovani tion in 30 ␮l were 6 pmol of each primer (for-
complex, Mundodi et al. (2002) have compared a L. ward: 5 -CGTGACGCCGGTGAAGAAT-3 ; reverse:
donovani strain and a L. chagasi (syn L. infantum) strain 5 -CGTGCACTCGGCCGTCTT-3 ), 4.5 nmol dNTPs,
and revealed at least five tandemly arranged genes. The 1 U Taq polymerase (Roche Diagnostics), 3 ␮l Buffer
last repeat of the cpb cluster was named cpbE for L. infan- 10× and either 100 pg of genomic DNA or 1 ␮l of
tum and cpbF for L. donovani. We used these two cpb DNA after InstaGeneTM extraction 200 ␮l or 1–5 ␮l of
copies to design a discriminative PCR for the L. dono- DNA extracted from clinical samples. Thirty cycles were
vani complex. Within a sample that was representative of necessary for amplification (denaturation 30 s at 94 ◦ C,
the genetic and clinical diversity of the L. donovani com- annealing 1 min at 62 ◦ C and elongation 1 min at 72 ◦ C),
plex, we evaluated the use of this PCR for discrimination followed by 10 min at 72 ◦ C. PCR amplification was also
of the two species. Other Leishmania and Trypanosoma tested with 50 pg of genomic DNA. The amplification
strains where included in this study. reactions were analyzed by agarose gel electrophoresis,
followed by ethidium bromide staining and visualization
2. Material and methods under UV light.

2.1. Parasites 3. Results

A sample of 32 strains that were representative of Thirty-two DNA samples corresponding to the strains
geographic and genetic diversity within the L. dono- of the L. donovani complex were amplified using spe-
vani complex was studied (Table 1). Ten parasite strains cific primers designed in this study. Optimal conditions
pertaining to other Leishmania species and to trypanoso- were found using 100 pg, but 50 pg could be used by
matids were used to test PCR specificity (Table 1). All the increasing the number of PCR cycles to 40. There is
Leishmania strains were typed using MLEE by the WHO no amplification when less than 50 pg of DNA was used
reference centres of Montpellier (MON) and London (with 30, 40 and 45 cycles) (data not shown). Two lengths
(LON). of amplification products were obtained, 702 bp for cpbE
and 741 bp for cpbF (Fig. 1). For the L. donovani and
2.2. Clinical samples L. archibaldi strains, only the cpbF copy was obtained,
whereas the L. infantum strains contained only cpbE,
Five clinical samples (spleen aspirates) were obtained except one strain. The L. infantum strain LIPA59 (a
in Uttar Pradesh from human patients with confirmed strain from Algeria isolated from the cutaneous form
VL (S. Sundar, Banaras Hindu University, Varanasi, of the disease) had a heterozygous pattern comprising
India). the two cpb copies E and F. This heterozygous pat-
tern has been obtained from different cultures of a given
2.3. Cell culture LIPA59 clone (obtained by micromanipulation) and also
from different clones. Thus, this pattern was not due
Promastigote cultures were maintained at 26 ◦ C by to a mixture of two strains or just to a contamination.
weekly sub-passages in RPMI1640 medium, buffered Furthermore, other results based on microsatellite geno-
with 25 mM HEPES, 2 mM NaHCO3 and supplemented typing and sequencing of other cp copies (cpa and cpb)
with 10% heat-inactivated foetal calf serum, 2 mM as well as lpg2, amastin and iunh, have confirmed the
glutamine, 100 U/ml penicillin and 100 ␮g/ml strepto- heterozygous status of this strain LIPA59 (Hide, 2004).
mycin. Cultures were harvested by centrifugation and The three Sudanese strains, originally typed by MLEE as
stored at −80 ◦ C until DNA extraction. L. infantum, presented a PCR product corresponding to
 M. Hide, A.-L. Bañuls / Acta Tropica 100 (2006) 241–245 243

Table 1
Leishmania strains used in this study
Species WHO code Zymodemea GenBankAC no.c

L. donovani complex
L. donovani MHOM/ET/67/HU3 MON-18 cpbF AY896783
L. donovani MHOM/ET/00/HUSSEN LON-42 cpbF AY896785
L. donovani MHOM/KE/67/MRC(L)3 LON-44 or 46 cpbF AY896786
L. donovani IMAR/KE/62/LRC-L57 MON-37 cpbF AY896788
L. donovani MHOM/SD/82/GILANI MON-30 cpbF AY896784
L. donovani MHOM/CN/00/WangJie1 MON-35 cpbF AY896787
L. donovani MHOM/IN/80/DD8 MON-2
L. donovani MHOM/IN/61/L13 n.d.
L. donovani MHOM/IN/82/Patna1 LON-41
L. donovani MHOM/SD/68/1S n.d.
L. donovani MHOM/SD/97/LEM3431 MON-30
L. donovani MHOM/SD/97/LEM3435 MON-30
L. donovani MHOM/SD/97/LEM3449 MON-18
L. donovani MHOM/SD/97/LEM3475 MON-18
L. archibaldi MHOM/SD/62/LRC-L61 MON-82
L. archibaldi MHOM/SD/93/GE MON-82
L. infantum MHOM/FR/78/LEM75 MON-1 cpbE AY896781
L. infantum MHOM/FR/82/LEM356 MON-33 cpbE AY896779
L. infantum MHOM/FR/87/LEM1098 MON-1 cpbE AY896776
L. infantum MHOM/FR/85/LEM716 MON-1 cpbE AY896777
L. infantum MHOM/FR/85/LEM663 MON-1 cpbE AY896780
L. infantum MHOM/ES/81/BCN1 MON-29 cpbE AY896778
L. infantum MHOM/MA/67/ITMAP263 MON-1 cpbE AY896782
L. infantum MHOM/DZ/82/LIPA59 MON-24 cpbE AY896790/cpbF AY896789
L. infantum MHOM/GR/2003/GH14 MON-1
L. infantum MHOM/GR/2004/GH24 MON-1
L. infantum MCAN/GR/2005/GD28 MON-1
L. infantum MHOM/TN/80/IPT1 MON-1
L. chagasi MCAN/BR/84/CO910 MON-1
L. chagasi MHOM/BR/84/M8270 MON-1
L. chagasi MHOM/BR/85/M9702 MON-1
L. chagasi MHOM/PA/78/WR285 MON-1 cpbE AY896791
Reference strainsb
L. tropica MHOM/SU/74/SAF-K27
L. major MHOM/SU/73/5ASKH
L. mexicana MNYC/BZ/62/M379
L. braziliensis MHOM/BR/75/M2904
L. guyanensis MHOM/BR/78M5378
L. lainsoni MHOM/BR/81/M6426
L. equatoriensis MCHO/EC/82/LSP1
L. tarentolae RTAR/IT/81/G8
Trypanosoma cruzi CL brener
T. brucei Jua
a Zymodemes were determined with the MLEE method by the WHO reference centres of Montpellier (MON) and London (LON).
b Ten reference strains of other Leishmania species and Trypanosoma were used for PCR specificity test.
c The cpbE and cpbF sequences submitted to GenBank.

cpbF as L. donovani. This PCR did not generate ampli- of 100 pg. These fragment lengths varied from 200 to
fication for other Leishmania species, representative of 450 bp (Fig. 1) and cannot be mistaken with the true cpb
the genetic diversity of the genus and for Trypanosoma products.
cruzi and T. brucei. Indeed, only some slight nonrepro- The PCR products have been sequenced for 15 strains
ducible patterns sometimes appeared for L. braziliensis, and the corresponding GenBank accession numbers are
L. guyanensis, L. lainsoni, L. tarentolae, T. cruzi and T. mentioned in Table 1. The difference between the two
brucei when 100 ng of DNA was used for PCR instead species was mainly due to a deletion of 39 nucleotides in
244 M. Hide, A.-L. Bañuls / Acta Tropica 100 (2006) 241–245

Fig. 1. Polymerase chain reaction using specific cpbEF primers. Amplification using 100 pg of genomic DNA was only obtained for L. donovani
strains (cpbF product, 741 bp) and L. infantum strains (cpbE product, 702 bp). L. infantum LIPA59 revealed both cpbE and cpbF products. There is
no amplification at these lengths for the other trypanosomatid species. On each side, molecular weight.

the mature domain of the cpbE sequences (Hide et al., in L. donovani complex (with or without mass culture) in
press). This deletion is responsible for the size polymor- the frame of epidemiological and taxonomic studies for
phism observed (702 bp for cpbE and 741 bp for cpbF). example.
The amplification using DNA from Indian VL patients PCR amplification revealed the presence of a cpbEF
did not produce a PCR product and thus, this PCR was heterozygous genotype for L. infantum LIPA59 isolated
not sensitive enough for diagnosis on the clinical samples from a human cutaneous lesion. Other results based
tested. on microsatellite genotyping and sequencing of other
cp copies (cpa and cpb) as well as lpg2, amastin and
4. Discussion iunh have suggested the hybrid status of this strain
LIPA59 (Hide, 2004). This strain might result from a
This study presents a discriminative PCR of the L. hybridization process between the two species L. infan-
donovani complex based on the cpb multigene family. tum and L. donovani due to a sexual recombination.
It discriminates between L. donovani and L. infantum Other hybrids have already been identified within the
species based on a difference in product length and does Leishmania genus as being between L. braziliensis and
not generate amplification for other trypanosomatids. L. panamensis/guyanensis (Bañuls et al., 1997; Belli et
The specificity of this PCR, in addition to its sensitiv- al., 1994), L. braziliensis and L. peruviana (Dujardin
ity (50 pg), demonstrates its usefulness in Leishmania et al., 1995) and L. major and L. arabica (Evans et al.,
typing. Furthermore, this PCR is more rapid than the 1987). However, this is the first description of a potential
classical MLEE method and does not need mass culture sexual recombination process between L. infantum and
because DNA extraction can be performed directly on L. donovani.
1 ml of primary culture using InstaGeneTM Matrix (Bio- The taxonomic status of strains such as L. donovani
Rad). However, this PCR is not sensitive enough for the MON30, initially typed as L. infantum by MLEE, was
diagnosis on clinical samples. This negative result could confirmed by this analysis (Oskam et al., 1998; Quispe
be due to an inhibition by spleenic constituents but this Tintaya et al., 2004; Zemanova et al., 2004). L. archibaldi
has not been tested in this study. Nevertheless, it appears and L. donovani revealed a common PCR pattern (cpbF,
very useful for the discrimination of strains within the 741 bp) and this diagnostic PCR could contribute to clar-
 M. Hide, A.-L. Bañuls / Acta Tropica 100 (2006) 241–245 245

ify the phylogenetic status of several taxa that continue to Evans, D.A., Kennedy, W.P.K., Elbihari, S., Chapman, C.J., Smith, V.,
be debated such as L. archibaldi (Jamjoom et al., 2004; Peters, W., 1987. Hybrid formation within the genus Leishmania?
Mauricio et al., 2001; Oskam et al., 1998; Zemanova et Parassitologia 29, 165–173.
Hide, M., 2004. Pathogenic variability with the Leishmania (Leish-
al., 2004) by the comparison of their cpbF sequences for mania) donovani complex, agent of visceral leishmaniasis.
example. Comparative study of biological and genetic characters and gene
expression. Ph.D. University of Montpellier II, 446 pp.
Conflict of interest Hide, M., Bañuls, A.L., Tibayrenc, M., 2001. Genetic heterogene-
ity and phylogenetic status of Leishmania (Leishmania) infantum
zymodeme MON-1: epidemiological implications. Parasitology
We have no potential conflicts of interest concerning 123, 425–432.
this manuscript. Hide, M., Bras-Gonçalves, R., Bañuls, A.L. Specific cpb copies within
the L. donovani complex. Evolutionary interpretations and poten-
Acknowledgements tial clinical implications in humans. Parasitology, in press.
Jamjoom, M.B., Ashford, R.W., Bates, P.A., Chance, M.L., Kemp, S.J.,
Watts, P.C., Noyes, H.A., 2004. Leishmania donovani is the only
MH is sponsored by CNRS and ALB by IRD. We cause of visceral leishmaniasis in East Africa; previous descrip-
gratefully thank I. Mauricio and F. Pratlong who have tions of L. infantum and “L. archibaldi” from this region are a
provided some Leishmania strains as well as G. Scho- consequence of convergent evolution in the isoenzyme data. Para-
nian and K. Soteriadou who provided some Leishmania sitology 129, 399–409.
Mauricio, I.L., Gaunt, M.W., Stothard, J.R., Miles, M.A., 2001. Genetic
DNA and C. Barnabe for DNA of T. brucei and T.
typing and phylogeny of the Leishmania donovani complex by
cruzi. We also thank S. Sundar and his lab who per- restriction analysis of PCR amplified gp63 intergenic regions. Par-
formed the PCR using DNA from Indian VL patients. asitology 122, 393–403.
The English version of this manuscript was revised by L. Mundodi, V., Somanna, A., Farrell, P.J., Gedamu, L., 2002. Genomic
Northrup. organization and functional expression of differentially regulated
cysteine protease genes of Leishmania donovani complex. Gene
282, 257–265.
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