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
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NOLDER AND OTHERS