86 research outputs found
Low-cost liquid medium for in vitro cultivation of Leishmania parasites in low-income countries
Background: Cutaneous Leishmaniasis (CL) induced by Leishmania aethiopica
has two clinical manifestations: ulcerating, self-healing CL and
non-ulcerating, non-healing CL. The grossly disfiguring multiple nodules
on the face and exterior surface of limbs during non-ulcerative CL are
sometimes misdiagnosed as other skin infections. Thus the need for
definitive and prompt laboratory diagnosis will be required. Identifying
Leishmania parasite by culture method is considered as a definitive
method for initiation of treatment and as an effective component of
leishmaniasis control methods. Recently the involvement of Fas (CD95)
and Tumor Necrosis Factor (TNF) Related Apoptosis Inducing Ligand (TRAIL)
induced apoptotic pathways were proposed to be involved in tissue
destruction and ulceration during L. major induced CL.
Aims: 1) to develop an alternative culture media that could minimize the
cost for culturing Leishmania from patient lesions.
2) to investigate if the expression of FasL and TRAIL differs in
ulcerating and non- ulcerative CL.
Methods: GALF-1 media was formulated in our lab and compared to RPMI 1640
medium and conventional Locke s semi solid media (LSSM) which is one of
the modifications of Novy-MacNeal-Nicolle (NNN) culture media.
Amastigotes transformation, cryopreservation, recovery of parasites, cost
and mass cultivation were analysed. Expression of Fas ligand (FasL),
TRAIL and apoptosis were assessed by immunohistology in human skin
biopsies from L. aethiopica induced ulcerative or non-ulcerative CL. FasL
and TRAIL blocking experiments were performed in a murine model of CL.
Results and discussion: GALF-1 is cheap and its ingredients available in
a low income country such as Ethiopia. GALF-1 was able to transform
amastigotes from Ethiopian patients samples and could be used to
cultivate promastigotes in large quantities. Cost analysis showed 80% to
95 % decreased costs as compared to conventional media. Promastigotes
cultured with GALF-1 could be cryopreserved in liquid nitrogen with
comparable re-culture potential to conventional media. Affordability of
diagnostic assays is a key issue for resource poor countries and the
possibility to cut the cost of the efficient culture method for diagnosis
through the use of inexpensive local formulated reagents could improve
the diagnosis of leishmaniasis in low income endemic countries.
More FasL expressing cells were detected in dermis of ulcerative CL as
compared to non-ulcerative CL and controls. TRAIL expression was higher
in ulcerative CL as compared to non-ulcerative CL and controls in both
epidermis and dermis. Increased dermal expression of FasL and TRAIL was
associated with ulcer formation during CL. This correlated with an
inhibition of the ulcerative process in a murine CL model during FasL and
TRAIL neutralisation.The mechanisms of the involvement of FasL and TRAIL
in ulceration was not elucidated and putative reason(s) for the
difference in dysregulation of apoptosis are discussed
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