A microculture technique for isolating live Leishmania parasites from peripheral blood of visceral leishmaniasis patients.

Abstract

Current procedures for diagnosing Leishmania parasites from patients involve invasive and dangerous tissue aspiration. We have developed a non-invasive and highly sensitive microculture method that can isolate parasites from the buffy coat of the patient's peripheral blood. The parasites were cultured in 96-well culture plates. Nineteen parasitologically proven visceral leishmaniasis (VL) patients were included in the study. Using this technique, we were able to isolate parasites from 16 (84%) samples. However, all 19 (100%) samples were positive on culture of splenic aspirates. We conclude that this technique is useful for the isolation and cryoconservation of parasites from patients' blood. This simple method could be tried as a first-instance alternative before other more sensitive procedures such as splenic aspirate; however, negative results should be confirmed by tests with higher sensitivity.

Full text

Page 1

Author's personal copy
2724, 911, Avenue Agropolis BP 64501, 34394 Montpellier Cedex
5, France. Tel.: +33 4 67 41 61 80; fax: +33 4 67 41 62 99.
E-mail address: hide@mpl.ird.fr (M. Hide).
Acta Tropica 102 (2007) 197–200
Short communication
A microculture technique for isolating live Leishmania parasites
from peripheral blood of visceral leishmaniasis patients
M. Hidea,∗, R. Singhb, B. Kumarb, A.L. Ba˜ nulsa, S. Sundarb
aG´ en´ etique et Evolution des Maladies Infectieuses, IRD/CNRS (UMR 2724), F-34394 Montpellier Cedex 5, France
bInfectious Diseases Research Laboratory, Department of Medicine, Institute of Medical Sciences, Banaras Hindu University,
6 SK Gupta, Nagar, Lanka, Varanasi 221005, India
Received 12 January 2007; received in revised form 18 April 2007; accepted 26 April 2007
Available online 1 May 2007
Abstract
Current procedures for diagnosing Leishmania parasites from patients involve invasive and dangerous tissue aspiration. We have
developed a non-invasive and highly sensitive microculture method that can isolate parasites from the buffy coat of the patient’s
peripheral blood. The parasites were cultured in 96-well culture plates. Nineteen parasitologically proven visceral leishmaniasis
(VL) patients were included in the study. Using this technique, we were able to isolate parasites from 16 (84%) samples. However,
all 19 (100%) samples were positive on culture of splenic aspirates. We conclude that this technique is useful for the isolation
and cryoconservation of parasites from patients’ blood. This simple method could be tried as a first-instance alternative before
other more sensitive procedures such as splenic aspirate; however, negative results should be confirmed by tests with higher
sensitivity.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Leishmania; Parasite isolation; Patient peripheral blood; Buffy coat; Non-invasive method
1. Introduction
Indian kala-azar or visceral leishmaniasis (VL)
causedbyLeishmaniadonovaniisapotentiallyfataldis-
ease. It is endemic in the eastern part of India and often
turns epidemic. Worldwide, nearly 500,000 new cases
of VL occur every year (Desjeux, 1992) with 100,000
cases of VL estimated to occur annually in India; the
state of Bihar accounts for majority of these 100,000
cases(Bora,1999).Althoughdemonstrationofparasites
in tissue smears is often used for the diagnosis of VL,
∗Corresponding author at: Laboratory GEMI, UMR CNRS/IRD
culture improves the specificity and sensitivity. Usually
tissuesfromeitherbonemarroworspleenareused;lym-
phadenopathy is rare in Indian VL. Sensitivity of tissues
from bone marrow is often very low, which makes it
unsuitableforparasitecultivation(ChulayandBryceson,
1983). Splenic aspiration is highly sensitive for the pur-
poses of parasite isolation, but it is a risky procedure
and may occasionally be associated with fatal haem-
orrhage (Sundar et al., 1991; Sundar and Rai, 2002).
Patients’ peripheral blood contains living amastigotes
(Lachaud et al., 2000; Osman et al., 1998), which could
be a very good source for diagnosis by culture. How-
ever, the isolation of parasites from the patients’ blood
may be difficult because of low parasitemia, and it is
not very well documented. In this paper, we describe a
microculture technique for the isolation of Leishmania
0001-706X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.actatropica.2007.04.015

Page 2

Author's personal copy
Inc.), 100?g/mL streptomycin (Sigma–Aldrich, Inc.),
1% l-glutamine (Lonza Ltd.), 0.5% gentamycin (Invit-
rogen Corporation) and 5% filtered human urine (donor:
Dr. M. Hide).
First, 10?L defibrinated rabbit blood was put in 30
wells of a 96-well culture plate (TPP®, Trasadingen,
198
M. Hide et al. / Acta Tropica 102 (2007) 197–200
parasites from the buffy coat of VL patients’ peripheral
blood. This culture method was thoroughly optimized
for the appropriate quantity of buffy coat and culture
medium. Parasite strains isolated using this method
have also been cultured and cryoconserved for further
studies.
2. Material and methods
2.1. Human blood samples
This study was conducted at the Infectious Dis-
eases Research Laboratory, Banaras Hindu University,
Varanasi, India. The Ethical Committee of Institute of
Medical Science, Banaras Hindu University, approved
the study. Informed written consent was obtained from
all patients. Nineteen parasitologically proven patients
(by microscopy of splenic smear) were selected for the
study.
2.2. Splenic smear
Smears were prepared by spreading splenic aspi-
rates on a glass slide. Smears were air-dried, fixed with
methanol and stained with a Giemsa solution. The par-
asite density score for pre- and post-treatment aspirates
was graded on the basis of the results of blinded micro-
scopic analysis using a conventional logarithmic scale
of 0, which indicated 0 parasites per oil-immersion
field (magnification, 1000) to +6, which indicated 1100
amastigotes per field (Chulay and Bryceson, 1983).
2.3. Micro-isolation
Five millilitres of peripheral blood were drawn from
the patients and centrifuged at 1000×g for 15min in
order to isolate the buffy coat. The Tobie’s blood-agar
(or NNN medium) was prepared by dissolving 15g
Bacto-tryptose(BDDifcoTM,FranklinLakes,NJ07417,
USA), 15g Bactoagar (BD DifcoTM), 4g NaCl, 5g
Na3PO4·12H2O and 0.4g KCl in 1L of distilled water.
The pH was adjusted to 7.6. The medium was auto-
claved at 120◦C for 15min prior to use. The RPMI
1640 medium (Lonza Ltd., Switzerland) was supple-
mentedwith20%heat-inactivatedfetalcalfserum(FCS)
(Lonza Ltd.), 100units/mL penicillin (Sigma–Aldrich,
Fig. 1. The 96-well plate disposition. In the 30 black wells, 10?L
defibrinated rabbit blood was completed with 100?L warmed NNN
medium. Then 50?L complete RPMI was added to each well. The
other wells (white) were filled with 250?L sterile water.
Switzerland) (Fig. 1); 100?L of warmed NNN medium
(40◦C) was added to these wells. Then 50?L of com-
plete medium was added to each well. With one 96-well
culture plate, 10 wells were prepared for each blood
sample and 3 samples were treated in the same plate.
All wells without culture were filled with sterile water
to avoid evaporation during incubation. Finally, 50?L
of buffy coat was inoculated in 10 wells and the 96-
well culture plate was sealed with parafilm and kept
in an incubator at 27◦C. In parallel, several wells were
inoculated with a tiny amount of splenic aspirate mate-
rial in order to compare both methods. Cultures were
examined on alternate days. After detection of posi-
tive culture, promastigotes were transferred in complete
medium. Cultures were examined every day under the
microscope.
2.4. Cryo-conservation
After observation of living parasites, the culture was
maintained for 1 or 2 weeks more (until the well was
full of parasites) and then inoculated in a culture tube
(Tube Nunclon, tissue culture, round polystyrene, radia-
tion sterilised, screw cap, 100mm×14mm, NuncTM)
containing 500?L of NNN medium (with 50?L of
defibrinated rabbit blood) and 250?L of complete
RPMI.Theculturewasslowlydilutedbyadding250?L
of medium every 2–3 days. After 2–3 weeks, the para-
sites were cryopreserved in nitrogen after addition of
33?L of DMSO in 1mL of culture or cultured in mass.
3. Results and discussion
Promastigotes were detected in 16 (84%) buffy coat
samples, whereas all 19 (100%) samples were posi-

Page 3

Author's personal copy
results should be confirmed by tests with higher sensi-
tivity.
Development of highly sensitive and specific diag-
nostic tools such as the direct agglutination test (DAT),
the rK39 strip test and polymerase chain reaction (PCR)
has made it highly likely that the gold standard par-
M. Hide et al. / Acta Tropica 102 (2007) 197–200
199
tive on splenic aspirate culture. The most important
difference observed in buffy coat and splenic aspirate
culture was that the time necessary to observe live par-
asites was longer in the buffy coat preparation than
in splenic aspirate culture. In general, positive cul-
tures were detected within 7–20 days using the buffy
coat, whereas only 5–10 days were required for splenic
aspirates. This observation might stem from relatively
low parasite density in peripheral blood. We also tried
to culture parasites using peripheral blood mononu-
clear cells (PBMCs), but we could not detect parasites
in any samples (data not shown). This last method
concentrates parasites in a lower volume but requires
more experiments and time. This lower efficiency has
already been pointed out by several authors (Riera et al.,
2004).
In the present study, we demonstrated that the blood
of Leishmania-infected persons can be used to diag-
nose visceral leishmaniasis and to isolate parasites. The
microculture technique developed is non-invasive and
has a significant advantage over the invasive and dan-
gerous splenic aspiration. Furthermore, our purpose in
using culture plates instead of culture tubes was to
reduce the quantity of culture medium and buffy coat.
Parasites grow better in 96-well plates because of the
lower volume of the two media and the proportion of
air to medium culture appears favourable for Leishma-
nia growth. Indeed, a large amount of air in the culture
environment is involved in a natural basification of the
RPMI, which is not favourable for parasite develop-
ment. We used sterile human urine along with fetal
calf serum, which have both been shown to be good
growth enhancers for Leishmania (Howard et al., 1991;
Singh et al., 2000). Other media such as normal saline
and Schneider insect medium have been tested on sev-
eral samples and have given positive cultures (data not
shown).
Moreover,thismethodmakesitpossibletoobtainlive
parasite strains, which can then be cultured for further
analysis or cryoconserved. In addition, this technique
would be also tested on isolate Leishmania parasites
from the blood of serologically positive, apparently
healthy endemic subjects in whom tissue aspiration
could not be done for ethical reasons. However, the
microculture technique should be considered as com-
plementary to other diagnostic protocols, and negative
asitology that involves invasive and dangerous tissue
aspiration will be replaced as the basis for the deci-
sion to treat VL patients. Hence, the method described
hereinishighlyrelevanttomeetingtheneedforparasites
for research purposes. Furthermore, antibody detec-
tion tests such as DiaMed-IT LEISH do not reveal the
parasite presence but only that the person has been
exposed to parasite. However, this method has lim-
ited utility for diagnostic purpose since faster tests are
available. Consequently, this method appears to be a
very harmless alternative to isolate parasite and could
be tried in a first instance before other, more sensitive
(but more invasive) procedures such as splenic aspi-
rate.
Conflict of interest
None.
Acknowledgements
This work was supported by a grant from the Indian
Council of Medical Research, India, and by the Institut
de Recherche pour le D´ eveloppement (IRD). MH and
ALB are supported by the IRD and the Centre National
delaRechercheScientifique(CNRS).Wearegratefulto
Jacques Dereure for useful advice. The English version
of this manuscript was revised by L. Northrup (English
Solutions).
References
Bora, D., 1999. Epidemiology of visceral leishmaniasis in India. Natl.
Med. J. India 12, 62–68.
Chulay, J.D., Bryceson, A.D., 1983. Quantitation of amastigotes of
Leishmania donovani in smears of splenic aspirates from patients
with visceral leishmaniasis. Am. J. Trop. Med. Hyg. 32, 475–
479.
Desjeux, P., 1992. Human leishmaniases: epidemiology and public
health aspects. World Health Stat. Q. 45, 267–275.
Howard, M.K., Pharoah, M.M., Ashall, F., Miles, M.A., 1991. Human
urinestimulatesgrowthofLeishmaniainvitro.Trans.R.Soc.Trop.
Med. Hyg. 85, 477–479.
Lachaud, L., Dereure, J., Chabbert, E., Reynes, J., Mauboussin, J.M.,
Oziol, E., Dedet, J.P., Bastien, P., 2000. Optimized PCR using
patient blood samples for diagnosis and follow-up of visceral
Leishmaniasis, with special reference to AIDS patients. J. Clin.
Microbiol. 38, 236–240.
Osman, O.F., Oskam, L., Zijlstra, E.E., el-Hassan, A.M., el-Naeim,
D.A., Kager, P.A., 1998. Use of the polymerase chain reaction to
assess the success of visceral leishmaniasis treatment. Trans. R.
Soc. Trop. Med. Hyg. 92, 397–400.
Riera,C.,Fisa,R.,Udina,M.,Gallego,M.,Portus,M.,2004.Detection
of Leishmania infantum cryptic infection in asymptomatic blood
donors living in an endemic area (Eivissa, Balearic Islands, Spain)

Page 4

Author's personal copy
200
M. Hide et al. / Acta Tropica 102 (2007) 197–200
by different diagnostic methods. Trans. R. Soc. Trop. Med. Hyg.
98, 102–110.
Singh, S., Mohapatra, D.P., Sivakumar, R., 2000. Successful replace-
ment of fetal calf serum with human urine for in vitro culture of
Leishmania donovani. J. Commun. Dis. 32, 289–294.
Sundar,S.,Kumar,K.,Singh,V.P.,Mahopatra,T.M.,1991.Diagnostic
lag period in kala-azar: test for early diagnosis needed. J. Assoc.
Physicians India 39, 651.
Sundar, S., Rai, M., 2002. Laboratory diagnosis of visceral leishmani-
asis. Clin. Diagn. Lab. Immunol. 9, 951–958.