Multiple hybrid genotypes of Leishmania (viannia) in a focus of mucocutaneous Leishmaniasis.
ABSTRACT The principal agent of mucocutaneous leishmaniasis (MCL) is the South American protozoan parasite Leishmania (Viannia) braziliensis. This organism is generally considered to be clonal, that is, it does not to undergo genetic exchange. Nevertheless, apparent hybrids between several Leishmania species have been reported in the New World and the Old World. When we characterized isolates of Leishmania (Viannia) from a single focus of cutaneous leishmaniasis (CL) and MCL, we found a remarkable phenotypic and genotypic diversity, with 12 zymodemes and 20 microsatellite genotypes. Furthermore, 26 of the 59 isolates were L. braziliensis/L. peruviana phenotypic hybrids that displayed 7 different microsatellite genotypes. A hybrid genotype was the only organism isolated from 4 patients with MCL. Thus hybrids must be included among the potential agents of MCL. Despite the propensity for clonality, hybrids are also an important feature of Leishmania (Viannia) and may give rise to epidemiologically important emergent genotypes.
- SourceAvailable from: Matthew B Rogers[show abstract] [hide abstract]
ABSTRACT: Although asexual reproduction via clonal propagation has been proposed as the principal reproductive mechanism across parasitic protozoa of the Leishmania genus, sexual recombination has long been suspected, based on hybrid marker profiles detected in field isolates from different geographical locations. The recent experimental demonstration of a sexual cycle in Leishmania within sand flies has confirmed the occurrence of hybridisation, but knowledge of the parasite life cycle in the wild still remains limited. Here, we use whole genome sequencing to investigate the frequency of sexual reproduction in Leishmania, by sequencing the genomes of 11 Leishmania infantum isolates from sand flies and 1 patient isolate in a focus of cutaneous leishmaniasis in the Çukurova province of southeast Turkey. This is the first genome-wide examination of a vector-isolated population of Leishmania parasites. A genome-wide pattern of patchy heterozygosity and SNP density was observed both within individual strains and across the whole group. Comparisons with other Leishmania donovani complex genome sequences suggest that these isolates are derived from a single cross of two diverse strains with subsequent recombination within the population. This interpretation is supported by a statistical model of the genomic variability for each strain compared to the L. infantum reference genome strain as well as genome-wide scans for recombination within the population. Further analysis of these heterozygous blocks indicates that the two parents were phylogenetically distinct. Patterns of linkage disequilibrium indicate that this population reproduced primarily clonally following the original hybridisation event, but that some recombination also occurred. This observation allowed us to estimate the relative rates of sexual and asexual reproduction within this population, to our knowledge the first quantitative estimate of these events during the Leishmania life cycle.PLoS Genetics 01/2014; 10(1):e1004092. · 8.52 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Chagas disease is a systemic pathology caused by Trypanosoma cruzi. This parasite reveals remarkable genetic variability, evinced in six Discrete Typing Units (DTUs) named from T. cruzi I to T. cruzi VI (TcI to TcVI). Recently newly identified genotypes have emerged such as TcBat in Brazil, Colombia and Panama associated to anthropogenic bats. The genotype with the broadest geographical distribution is TcI, which has recently been associated to severe cardiomyopathies in Argentina and Colombia. Therefore, new studies unraveling the genetic structure and natural history of this DTU must be pursued. We conducted a spatial and temporal analysis on 50 biological clones of T. cruzi I (TcI) isolated from humans with different clinical phenotypes, triatomine bugs and mammal reservoirs across three endemic regions for Chagas disease in Colombia. These clones were submitted to a nuclear Multilocus Sequence Typing (nMLST) analysis in order to elucidate its genetic diversity and clustering. After analyzing 13 nuclear housekeeping genes and obtaining a 5821 bp length alignment, we detected two robust genotypes within TcI henceforth named TcIDOM (associated to human infections) and a second cluster associated to peridomestic and sylvatic populations. Additionaly, we detected putative events of recombination and an intriguing lack of linkage disequilibrium. These findings reinforce the emergence of an enigmatic domestic T. cruzi genotype (TcIDOM), and demonstrates the high frequency of recombination at nuclear level across natural populations of T. cruzi. Therefore, the need to pursue studies focused on the diferential virulence profiles of TcI strains. The biological and epidemiological implications of these findings are herein discussed.BMC Genetics 09/2013; 14(1):96. · 2.81 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Visceral leishmaniasis (VL), a widely distributed systemic disease caused by infection with the Leishmania donovani complex (L. donovani and L. infantum), is almost always fatal if symptomatic and untreated. A rapid point-of-care diagnostic test for anti-Leishmania antibodies, the rK39-immunochromatographic test (rK39-ICT), has high sensitivity and specificity in South Asia but is less sensitive in East Africa. One of the underlying reasons may be continent-specific molecular diversity in the rK39 antigen within the L. donovani complex. However, a second reason may be differences in specific IgG anti-Leishmania levels in patients from different geographical regions, either due to variable antigenicity or immunological response. We determined IgG titres of Indian and Sudanese VL patients against whole cell lysates of Indian and Sudanese L. donovani strains. Indian VL patients had significantly higher IgG titres against both L. donovani strains compared to Sudanese VL patients (p<0.0001). Mean reciprocal log10 50% end-point titres (1/log10t50) were i) 3.80 and 3.88 for Indian plasma and ii) 2.13 and 2.09 for Sudanese plasma against Indian and Sudanese antigen respectively (p<0.0001). Overall, the Indian VL patients therefore showed a 46.8-61.7 -fold higher mean ELISA titre than the Sudanese VL patients. The higher IgG titres occurred in children (<16 years old) and adults of either sex from India (mean 1/log10t50: 3.60-4.15) versus Sudan (mean 1/log10t50: 1.88-2.54). The greatest difference in IgG responses was between male Indian and Sudanese VL patients of ≥ 16 years old (mean 1/log10t50: 4.15 versus 1.99 = 144-fold (p<0.0001). Anti-Leishmania IgG responses among VL patients in Sudan were significantly lower than in India; this may be due to chronic malnutrition with Zn(2+) deficiency, or variable antigenicity and capacity to generate IgG responses to Leishmania antigens. Such differential anti-Leishmania IgG levels may contribute to lower sensitivity of the rK39-ICT in East Africa.PLoS Neglected Tropical Diseases 02/2014; 8(2):e2675. · 4.57 Impact Factor
MULTIPLE HYBRID GENOTYPES OF LEISHMANIA (VIANNIA) IN A FOCUS OF
DEBBIE NOLDER,* NORMA RONCAL, CLIVE R. DAVIES, ALEJANDRO LLANOS-CUENTAS, AND
MICHAEL A. MILES
Department of Infectious & Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom;
Instituto de Medicina Tropical “Alexander von Humboldt”, Universidad Peruana Cayetano Heredia (UPCH), Lima, Peru
Leishmania (Viannia) braziliensis. This organism is generally considered to be clonal, that is, it does not to undergo
genetic exchange. Nevertheless, apparent hybrids between several Leishmania species have been reported in the New
World and the Old World. When we characterized isolates of Leishmania (Viannia) from a single focus of cutaneous
leishmaniasis (CL) and MCL, we found a remarkable phenotypic and genotypic diversity, with 12 zymodemes and 20
microsatellite genotypes. Furthermore, 26 of the 59 isolates were L. braziliensis/L. peruviana phenotypic hybrids that
displayed 7 different microsatellite genotypes. A hybrid genotype was the only organism isolated from 4 patients with
MCL. Thus hybrids must be included among the potential agents of MCL. Despite the propensity for clonality, hybrids
are also an important feature of Leishmania (Viannia) and may give rise to epidemiologically important emergent
The principal agent of mucocutaneous leishmaniasis (MCL) is the South American protozoan parasite
Leishmaniasis is a major public health problem in much of
Latin America, where Leishmania of the subgenus Leishma-
nia are agents of visceral leishmaniasis (VL), cutaneous leish-
maniasis (CL), and diffuse cutaneous leishmaniasis (DCL).
The subgenus L. (Viannia), which is restricted to the New
World, causes CL and metastatic mucocutaneous leishmania-
In Peru, both CL and MCL are endemic. Leishmania (Vi-
annia) braziliensis and L. (V.) peruviana are most frequently
associated with CL, although L. (V.) guyanensis, L. (V.) lain-
soni and L. (Leishmania) amazonensis have also been re-
ported.1MCL is attributed to L. braziliensis. However, le-
sions from L. braziliensis and L. peruviana are not distinct in
the early stages of CL, and species have often been incrimi-
nated on the basis of known geographical range, with L. pe-
ruviana found mostly in the western Andes and inter-Andean
valleys and L. braziliensis occurring predominantly at lower
altitudes in the Amazonian region. Mucosal leishmaniasis
(ML), with involvement of the mucosae by contiguity from a
primary lesion, has been described for all species causing CL
in Peru.1However, ML is distinct from MCL, which involves
metastatic spread to mucosal sites some time after a primary
infection. Control of MCL depends predominantly on passive
or active case finding, diagnosis, and effective treatment.2
Dogs are commonly infected with L. braziliensis and/or L.
peruviana, and they may act as a peridomestic reservoir.3,4
The sylvatic reservoir hosts of L. peruviana and L. braziliensis
are incompletely known, although terrestrial small rodents
have been implicated for some strains.4,5
Where the Amazonian forest and Andean regions meet, as
in the Department of Huánuco, both L. braziliensis and L.
peruviana may occur sympatrically. Leishmania (Viannia) iso-
lates that appear to be hybrids between L. braziliensis and L.
peruviana have been reported from this region of Peru.6
Herein we describe the phenotypes (obtained by multilocus
enzyme electrophoresis, MLEE), and genotypes (obtained by
microsatellite multilocus typing, MLMT) of 59 isolates from
the Department of Huánuco. The results show that putative
hybrid phenotypes and genotypes are common. A remarkable
degree of diversity is revealed, suggesting that propagation is
not entirely clonal and indicative of some form of genetic
exchange in the Huánuco L. (V.) population.
MATERIALS AND METHODS
Isolate collection. Leishmania were isolated in 1994–1995
from 45 humans and 14 dogs, from cutaneous lesions on the
ear or nose, in villages around Huánuco City (2000–3000 m
above sea level). Eleven patients had MCL, and 34 had CL.
Full ethical permission was given by the Universidad Peruana
Cayetano Heredia with informed consent obtained from hu-
man subjects for parasite diagnosis. Households in these areas
routinely keep dogs, donkeys, pigs, and chickens. A full list of
the isolate codes is available from the authors upon request.
Isolates were phenotyped and genotyped against the fol-
lowing L. (V.) reference strains: L. (V.) braziliensis (MHOM/
BR/84/LTB300); L. (V.) peruviana (MHOM/PE/94/LC1152;
MHOM/PE/84/LC26; MHOM/PE/84/LC39); L. (V.) pana-
mensis (MHOM/PA/71/LS94); L. (V.) guyanensis (MHOM/
BR/75/M4147); L. (V.) shawi (MHOM/BR/94/M15065); L.
(V.) lainsoni (MHOM/BR/81/M6426); and L. (V.) sp. n.7
Isolation and in vitro cultivation of parasites. Reference
strains were retrieved from liquid nitrogen storage onto bi-
phasic 4N blood slopes.8Peruvian stocks were dispatched
from Lima on 4N blood slopes and were passaged onto fresh
slopes and incubated at 23°C upon receipt. Isolates from this
first-passage culture were stored under liquid nitrogen; sub-
sequent passages were kept to a minimum to reduce culture
selection of parasite strains. Promastigote cultures were ex-
panded in alpha-modified minimal essential medium (Sigma-
Aldrich Ltd., Gillingham, Dorset, UK) supplemented with
10% heat-inactivated fetal bovine serum, 50 ?g/mL gentami-
cin, 30 mM NaHCO3, 40 mM HEPES, 20 mM D-glucose, 4
mM L-glutamine, 10 ?M hemin, 30 ?M adenine, 10 ?M folic
acid, and 10 ?M D-biotin (all supplements from Sigma Chemi-
* Address correspondence to Debbie Nolder, Department of Infec-
tious & Tropical Diseases, London School of Hygiene & Tropical
Medicine, Keppel Street, London WC1E 7HT, United Kingdom.
Am. J. Trop. Med. Hyg., 76(3), 2007, pp. 573–578
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene
cal Co., Gillingham, Dorset, UK). Enzyme lysates were pre-
pared from logarithmic-phase bulk cultures according to the
method of Evans and others.8
Isoenzyme electrophoresis. Diversity of the Leishmania
isolates was initially analyzed by MLEE using thin-layer
starch gels.9The enzymes applied were mannose phosphate
isomerase (MPI, EC 220.127.116.11); glucose phosphate isomerase
(GPI, EC 18.104.22.168); proline dipeptidase (PEPD, EC 22.214.171.124);
phosphoglucomutase (PGM, EC 126.96.36.199); nucleoside hydro-
lase using 2 different substrates, inosine (NHi, EC 188.8.131.52; 2
loci: NHi1 and NHi2) and deoxyinosine (NHd, EC 3.2.2.x);
6-phosphogluconate dehydrogenase (6PGD, EC 184.108.40.206);
glucose-6-phosphate dehydrogenase (G6PD, EC 220.127.116.11); es-
terase (ES, EC 18.104.22.168); aspartate aminotransferase (ASAT,
EC 22.214.171.124); and alanine aminotransferase (ALAT, EC
Microsatellite genotyping and DNA sequencing. Subse-
quent analyses involved multilocus microsatellite typing
(MLMT)10and DNA sequencing. Micosatellites AC01,
AC16, and AC5211were amplified from promastigote geno-
mic DNA. The micosatellites were amplified with primers
AC01F and AC01R-FAM (GAGAGGCCACCAGACACG-
TCAGCACAC and CCCCCTTCCTTCGCCTTCAACAC-
CTTTAC, respectively), AC16F and AC16R-TET (CTTCT-
TCTCATGCTGCACGGTCTCCTCCTT and CCATGG-
GCGGGCTTGTTTCGTTACTTTTTA, respectively), and
AC52F and AC52R-HEX (CCACCGCCGGCTTCACTAC
and GCGGCAATCGTCTGGCTAAA, respectively). Re-
verse primers were fluorophore-labeled as indicated (Perkin-
Elmer, Beaconsfield, UK). Amplification reactions were car-
ried out according to a protocol modified from that of Russell
and others.11PCR amplification for each sample was done in
a 10 ?L reaction mix containing 10× NH4reaction buffer (160
mM (NH4)2SO4, 670 mM Tris-HCl, pH 8.8, 0.1% Tween-20
[Bioline, London, UK]), 1 mM (AC01, AC52) or 2 mM
(AC16) MgCl2, 0.2 mM each of dATP, dCTP, dGTP, and
dTTP (Pharmacia LKB, Upsala, Sweden), 5 pmol of each
primer, 0.5% formamide (v/v), 1 U of Taq DNA polymerase
(Bioline), and 25 ng of genomic DNA. PCR amplification was
carried out in microtiter plates with sealed lids using the
heated-lid option in an MJ Research PTC-200 Peltier ther-
mocyler (Genetic Research Instrumentation Ltd., UK) using
the following parameters: 35 cycles of 95°C for 30 s, 62°C
(AC01 and AC52) or 60°C (AC16) for 30 s, 72°C for 1 min,
followed by a final extension period of 10 min at 72°C. The
multiplexed microsatellite products were sized on an ABI 377
automated sequencer by Genescan® and Genotyper® soft-
ware (Applied Biosystems, Warrington, UK).
The AC01 products were sequenced by dye-terminator
cycle sequencing and aligned using Sequence Navigator (Ap-
Population genetics analysis. Resultant data from MLEE
and MLMT were tested for genetic recombination and seg-
regation using five population-genetics analyses: Hardy-
Weinberg (HW) equilibrium,12fixation index (Fis),12D? in-
dex,13, R2index,13and index of association (IA).14
The isoenzyme phenotypes of the isolates were diverse.
Twelve zymodemes (LON217-228) were encountered among
the 59 Huánuco stocks by MLEE. One human isolate was
identified as L. lainsoni, and 3 dog isolates as L. (V.) sp. n.7.
For the remaining 55 isolates (Table 1), the enzymes GPI,
G6PD, ASAT, and ALAT were monomorphic while MPI,
PEPD, PGM, NHi, NHd, 6PGD, and ES were polymorphic.
The 55 isolates were initially classified according to their de-
fining MPI profile, diagnostic for L. braziliensis and L. peru-
viana; 25 isolates were L. braziliensis (inferred MPI genotype
1/1), 4 were L. peruviana (2/2), and 26 had a double-banded
MPI profile indicative of L. braziliensis/L. peruviana hybrids
(1/2). Mixed cultures were excluded both by the occurrence of
the same phenotype in biologic clones6and by distribution of
band intensities, including the presence of the classic triple-
banded hybrid phenotype (with a central band of greater in-
tensity) for dimeric enzymes.6,9All putative hybrids had a
triple-banded profile for the dimeric enzyme NHd. In con-
trast to the L. braziliensis/L. panamensis hybrids from Nica-
ragua,9the Huánuco hybrids did not have the same overall
phenotype (Tables 1 and 2).
Twenty multilocus microsatellite genotypes were identi-
fied, including 6 different multilocus genotypes among iso-
lates from MCL lesions (Tables 1 and 2). Double peaks indi-
cated heterozygosity. Twenty-three L. braziliensis/L. peruvi-
ana hybrids were heterozygous at the AC01 locus, as were
many L. braziliensis. AC01 DNA sequence was obtained for
all 55 isolates that generated a PCR product for the AC01
Zymodeme and microsatellite genotypes of 55 Leishmania (Viannia)
isolates, Huánuco, Peru
12 (2) (V)
Bold shows the phenotypes (zymodemes) and the multilocus microsatellite genotypes
associated with mucosal disease (MCL); X ? not amplifiable; ND ? not done; NS ? not
scorable by Genotyper®, *Bold in parentheses shows numbers of isolates from MCL, roman
numerals (I, V) show number of isolates from dogs. Reference strains used in phenotyping
and genotyping were L. (V.) braziliensis (MHOM/BR/84/LTB300); L. (V.) peruvian
(MHOM/PE/94/LC1152; MHOM/PE/84/LC26; MHOM/PE/84/LC39); L. (V.) panamensis
(MHOM/PA/71/LS94); L. (V.) guyanensis (MHOM/BR/75/M4147); L. (V.) shawi (MHOM/
BR/94/M15065); L. (V.) lainsoni (MHOM/BR/81/M6426); and L. (V.) sp. n.7(ISQU/BR/86/
NOLDER AND OTHERS
locus. In most cases, the number of dinucleotide repeats from
sequence analysis correlated exactly with allele size by Geno-
typer®. When isolates were scored as heterozygous, the su-
perimposed downstream sequences were clearly interpretable
as 2 alleles differing by number of repeats. For example, all L.
braziliensis isolates scored using Genotyper® as 227/231 were
found to have overlapping sequence reads that could be in-
terpreted as 2 alleles differing by 2 repeats (i.e., 8 and 10),
giving allele sizes that varied by 4 base pairs. Only 6 of the L.
braziliensis/L. peruviana hybrids were heterozygous at the
unlinked AC16 locus. The complexity of AC52 profiles only
allowed limited allele scoring.
Five population-genetics analyses, Hardy-Weinberg (HW)
equilibrium,12fixation index (Fis),12D? index,13, R2index,13
and index of association (IA),14were applied to the data for
58 isolates, including (due to apparent sharing of alleles) L.
(V.) sp. n.7but excluding L. lainsoni. The results were as
1. Four of 10 polymorphic enzyme loci (MPI, ES, PEPD, and
NHd) showed no significant deviation from HW equilib-
rium (P > 0.05, HW exact test), whereas 4 enzyme loci
(PGM, GPI, G6PD, NHi1) and both microsatellite loci
(AC01, AC16) deviated from HW equilibrium (NHi2 and
ASAT were monomorphic, ALAT and 6GPD had null
2. Excess heterozygosity was indicated by negative Fis values
at 3 of 10 polymorphic loci MPI (−0.133), NHi (−0.425),
NHd (−0.281), and AC01 (−0.288), whereas 4 loci showed
deficits of heterozygosity, 3 of which were entirely ho-
3. D? indices were calculated for pairwise combinations of
loci, with “1” denoting complete linkage and “0” indicating
no linkage.13Calculations were based on maximum-
likelihood estimates of gametic frequencies.13Reference
to the L. major genome sequencing project indicates at
least 8 different chromosomal assignments, although the
loci are not yet mapped for L. (Viannia). Seven out of 13
pairwise combinations showed a value of D? < 0.7. Three of
these pairwise values (MPI × NHi1, AC16 × NHi1, and
AC16 × NHd) were statistically significant (P ? 0.01, Q
test).13Corresponding r2values of linkage disequilibrium
were lower, as is normal for this index.13
4. The Maynard Smith index of association, IA, which as-
sesses linkage disequilibrium over all loci, was calculated.
IAvalues for all enzyme loci (0.59) and for all loci (0.97)
did not indicate panmixia (IA? 0). Values are comparable
to those obtained with a smaller number of loci for Try-
panosoma brucei populations exhibiting epidemic clonal-
In summary, overall the results from these tests indicated that
the Huánuco L. (Viannia) population diverges from clonality
or linkage disequilibrium. Firstly, the partial lack of signifi-
cant deviation from HW equilibrium and, secondly, the sig-
nificant D? values imply that some form of genetic exchange
has occurred among the Huánuco L. (Viannia) population.
The trypanosomatids that cause human diseases (trypano-
somiasis and leishmaniasis) were for many years considered
to be incapable of genetic exchange, with reproduction con-
fined to binary fission, in which one parental form divides to
produce two identical offspring. Sexual dimorphism is not
apparent, and chromosomes do not condense and therefore
cannot be visualized. As a result, meiosis and mitosis cannot
be observed directly.
Perceptions that T. brucei and Trypanosoma cruzi are en-
tirely clonal have changed dramatically. With the aid of drug-
resistance markers, genetic exchange was demonstrated to
occur in the tsetse fly salivary glands, both within and be-
tween the T. brucei subspecies.16The mechanism appears to
be Mendelian, with occasional aneuploid progeny.17Depen-
dent on the locality under study, the population genetics of T.
brucei ranges from panmixia in some undisturbed natural
hosts and habitats through epidemic clonality, to true clonal-
ity.15Among T. cruzi populations, phylogenetic analysis re-
vealed genetic exchange18and hybrids were produced experi-
mentally in the laboratory.19The T. cruzi experimental hy-
brids were derived from mammalian cells, not from the
triatomine bug vector19(although this does not exclude oc-
currence of genetic exchange within the vector). The mecha-
nism of hybridization in T. cruzi, involving fusion, genome
erosion, and recombination, was unusual but compatible with
hybrid genotypes and the extensive range of DNA content
found among natural populations. Heitman20has drawn at-
tention to striking parallels between T. cruzi and the fungus
Candida albicans: cell fusion in C. albicans yields tetraploid
progeny, which in appropriate growth conditions undergo
random chromosome loss to revert to diploidy.21
Similarly, perceptions of Leishmania as entirely clonal have
been questioned by reports of several instances of naturally
occurring hybrid strains, especially from the New World. On
the basis of phenotypic and genotypic markers, hybrids have
been described between L. braziliensis and L. panamensis in
Nicaragua9; between L. braziliensis and L. guyanensis in Ven-
ezuela22; between L. braziliensis and L. guyanensis in Ecua-
dor23; and between L. braziliensis and L. peruviana in Peru.6
In the Old World, L. major/arabica hybrids were described.24
Putative parental and hybrid phenotypes of the L. donovani
complex (L. donovani; “L. archibaldi”) occur, sympatrically
in East Africa, and sequencing of housekeeping genes encod-
ing enzymes shows mosaic characters across such strains.25
Preliminary genetic analysis suggests that genetic exchange
may occur among L. tropica populations in the Middle East.26
Most recently, genetic hybrids between L. infantum and L.
major have been described, from immunocompromised pa-
tients in Portugal.27Mixed Leishmania infections occur in
natural hosts and in vectors, and infections in mammals may
Summary of zymodemes and multilocus microsatellite genotypes
No. of isolates
No. of zymodemes
(10 loci) [MCL
Minimum+ no. of
(3 loci) [MCL associated]
L. b/pe hybrids
L. (V.) sp. n.
* 1 from dog; †10 from dog; §all from dog; +X and NS are not considered distinct geno-
types here (see Table 1).
MULTIPLE HYBRID GENOTYPES OF LEISHMANIA VIANNIA
last for decades, providing ample opportunities for interac-
tions between distinct genotypes. Overall, this is substantial
circumstantial evidence that an extant mechanism of genetic
exchange remains to be described for Leishmania, with epi-
demiologic implications, for example, in the spread of emer-
gent strains or drug resistance.
L. peruviana was recorded in the Department of Huánuco
prior to the epidemic from which the isolates in this study
were derived. However, prior to this epidemic, CL was sel-
dom encountered and no cases of MCL were recorded
(Llanos-Cuentas, unpublished data). It is likely that introduc-
tion of L. braziliensis6resulted in the increased prevalence of
CL, and outbreak of MCL. L. braziliensis may have been
introduced by human immigration from another region or by
human or canine intrusion into an unidentified sylvatic trans-
The 59 Huánuco isolates analyzed here showed a remark-
able degree of isoenzyme diversity considering that they orig-
inated from such a small geographical area. L. guyanensis and
L. amazonensis, previously reported from Peru,1were not
identified. However, 4 species—L. peruviana, L. braziliensis,
L. lainsoni, and L. (V.) sp. n.7—were found to occur sym-
patrically in Huánuco. In addition, L. braziliensis/L. peruvi-
ana phenotypic hybrids were common and almost as abun-
dant as L. braziliensis. This local prevalence of hybrid strains
is reminiscent of the predominance of the genetic hybrids of
T. cruzi among human cases of Chagas disease in Paraguay
and adjacent regions.28The gene encoding the enzyme MPI
has recently been sequenced from both L. braziliensis and L.
peruviana, and phenotypic differences have been shown to
correspond with a single nucleotide polymorphism (SNP),
changing a threonine to an arginine, which has been used as
the basis of a PCR identification assay.29It would be of in-
terest to confirm that the current L. braziliensis, L. peruviana,
and L. braziliensis/L. peruviana hybrid isolates, and those
from a wider geographical range, conform to the predicted
It was surprising to find 4 MLEE phenotypes and 7 micro-
satellite genotypes among the L. braziliensis/L. peruviana hy-
brids. This might be a consequence of the rapid evolution of
microsatellites, or it could be consistent with the occurrence
of more than one hybridization event. It is very unlikely that
these genotypes are explicable by mutation, with no hybrid-
ization event. By analogy, multilocus sequence typing
(MLST) of the L. donovani complex has revealed multiple
heterozygous sites within a gene and at several loci, with shar-
ing of alleles within and across genetic groups: recombina-
tion,25,30rather than mutation,31is considered to be the most
The majority (12) of the L. braziliensis/L. peruviana hy-
brids had a single genotype (Table 1). The expansive clonal
propagation of one of the putative hybrid genotypes suggests
that a hybrid agent has emerged with increased fitness rela-
tive to the parental strains, although a neutral event, such as
a population bottleneck, unlikely in view of the diversity
within the population, may also have been involved. One
comparison of promastigote growth rates found no evidence
that L. braziliensis/L. peruviana hybrids had an enhanced
growth rate in vitro.32. Nevertheless, it would be of interest to
compare the metastatic potential of the hybrid and nonhybrid
genotypes in the hamster model of MCL33or perhaps in the
mouse ear model for dissemination.34The occurrence of L.
braziliensis/L. peruviana hybrids in dogs and humans (Tables
1 and 2) from the same area indicates that both are exposed
to the same infective sand fly population. This suggests that
dogs might act as a reservoir host and enhance propagation of
the emergent hybrid genotype.
Six zymodemes and at least 6 microsatellite genotypes were
isolated from patients with MCL. Isolation of L. peruviana
from a single case of MCL is an interesting finding but, in the
absence of more cases, must be interpreted with caution. Pre-
viously in Peru, a single case of mucosal leishmaniasis (ML)
was attributed to L. peruviana.1Four of the MCL patients
yielded a hybrid isolate, with no evidence of mixed infection,
so patients carrying such isolates must be considered at risk of
developing MCL. Thus, the finding of unequivocal genotypic
markers for isolates that carry the risk of progression to MCL
is still an elusive goal.35
As mentioned above, MLST applied to the L. donovani
complex has already revealed the genetic basis of MLEE and
provided a higher resolution approach to resolving genetic
groups and to understanding relationships between them.25,30
MLMT has given even higher resolution of intraspecific
population structure.10Diversity of the subgenus L. (Viannia)
is seen in some endemic foci,36although the level observed in
Huánuco is extraordinary. Genetic diversity has also been
recorded from individual patients.37Furthermore, it is appar-
ent that there is some overlap of boundaries between the
perceived species of the subgenus L. (Viannia). In combina-
tion, MLST and MLMT will be a powerful approach to un-
derstanding the complex molecular epidemiology and popu-
lation genetics of L. (Viannia) and for further investigation of
the extent of genetic exchange in natural or experimental
populations. For the future MLMT of L. (Viannia), a much
wider panel of microsatellite markers is required, and such a
panel is in preparation (R. Oddone, G. Schönian, and K.
Kuhls, personal communication and unpublished data). In lo-
calities where L. (Viannia) species overlap, we anticipate the
discovery and possible emergence of other human infective
hybrid genotypes, some with potential to generate severe dis-
Received May 10, 2006. Accepted for publication October 31, 2006.
Acknowledgments: The authors thank Rachel Gregory (LSHTM) for
technical assistance, David Conway (LSHTM), Helen Roberts (Uni-
versity College, London), and Isabel Mauricio (LSHTM) for helpful
discussions. Dr. R. Naiff (Instituto Nacional de Pesquisas Amazonas,
Manaus, Amazonas, Brazil) and Prof. J. Shaw (Instituto Evandro
Chagas, Belém, Pará, Brazil) generously provided some of the refer-
ence strains used in this study.
Financial Support: This work was supported by European Commis-
sion International Scientific Co-operation (grant no. C11-CT93-0036-
NR), the Sir Halley Stewart Trust, and the Wellcome Trust.
Authors’ addresses: Debbie Nolder, Clive R. Davies, and Michael A.
Miles: Department of Infectious & Tropical Diseases, London
School of Hygiene & Tropical Medicine, Keppel Street, London
WC1E 7HT, United Kingdom, Telephone: +44 (0)20 7927 2427, Fax:
+44 (0)20 7637 0268, E-mails: firstname.lastname@example.org,
email@example.com, and firstname.lastname@example.org. Norma
Roncal: Instituto de Medicina Tropical “Alexander von Humboldt”,
Universidad Peruana Cayetano Heredia, AP5045, Lima 100, Peru,
E-mail: email@example.com. Alejandro Llanos-Cuentas: Facultad
de Salud Pública y Administración, Carlos Vidal Layseca, Univer-
sidad Peruana Cayetano Heredia, AP5045, Lima 100, Peru, Tele-
phone: +511 482 7739/381 4100, Fax: +511 382 0338, E-mail:
NOLDER AND OTHERS
Reprint requests: Debbie Nolder, Malaria Reference Laboratory,
London School of Hygiene & Tropical Medicine, Keppel Street, Lon-
don WC1E 7HT, United Kingdom.
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