14th ICID - Poster Abstracts - International Society for Infectious ...

When citing these abstracts please use the following reference:
Author(s) of abstract. Title of abstract [abstract]. Int J Infect Dis 2010;14S1: Abstract number.
Please note that the official publication of the International Journal of Infectious Diseases 2010, Volume 14, Supplement 1
is available electronically on http://www.sciencedirect.com
Final Abstract Number: 75.043
Session: Diagnostics
Date: Friday, March 12, 2010
Time: 12:30-13:30
Room: Poster & Exhibition Area/Ground Level
Type: Poster Presentation
Molecular diagnosis of American tegumentary leishmaniasis with minimally invasive samples
B. Valencia 1 , N. Veland 1 , J. Arevalo 1 , A. Llanos-Cuentas 1 , J.-C. Dujardin 2
1 Institute of Tropical Medicine "Alexander von Humboldt", Lima, Peru, 2 Instituut voor Tropische
Geneeskunde, Antwerp, Belgium
Background: American Tegumentary Leishmaniasis (ATL) is endemic in nearly 65% of Peru.
Even when diagnosis is routinely done by direct examination, its sensitivity can be easily affected
by several factors such as disease duration, superinfection and appropriate sampling. The last
one is an important factor especially due to patient tolerability (particularly in facial lesions or
children) and to an adequate training.
Methods: Diagnosis of ATL was evaluated in 40 lesions by 3 methods: direct examination, culture
and PCR. PCR was performed in lymphatic secretion and dermal scrapings. Montenegro Skin Test
(MST) was not performed in all cases. Lesions were cleaned with hydrogen peroxide and then
lymphatic secretion was taken with microhematocrit capillary tubes. Dermal scrapings were taken
with two sterile lancets: one for PCR and the other one for direct examination. Culture was done by
the aspiration method. DNA was isolated by the phenol-chloroform-isoamilic alcohol method and
precipitated with ethanol and 3M sodium acetate. The kinetoplast DNA (kDNA) PCR was performed
with 0.5 U of HotStar Taq DNA Polymerase (QIAGEN), with a set of primers targeting the
Leishmania kDNA minicircles: MP1-L (fwd) 5’-TACTCCCCGACATGCCTCTG-3’ and MP3-H (rev)
5’-GAACGGGGTTTCTGTATGC-3’. Another set of primers targeting human beta-hemoglobin gene
was used in the same reaction tube as an internal amplification control: HBBL (fwd) 5’-
GGCAGACTTCTCCTCAGGAGTC-3’ and HBBR (rev) 5'-CTTAGACCTCACCCTGTGGAGC-3’.
Results: Of the 40 suspected ATL lesions, 39 were confirmed by at least one of the three
conventional diagnostic methods used here. Only one lesion was negative to all methods, but
MST was very strong, thereby supporting the diagnosis of ATL. The sensitivity of every method
was as follows: direct examination 75%, culture 50%, PCR of dermal scrapings 95% and PCR of
lymphatic secretion 90%. The DNA obtained from both PCR samples was enough for further
Leishmania species identification. Human DNA obtained from dermal scrapings was more
abundant in comparison to lymphatic samples.
Conclusion: PCR from dermal scrapings and lymphatic secretion have higher sensitivity in
comparison to traditional procedures. Lymphatic secretion has less human DNA and this can
improve the species typing process. We aim to show that diagnosis of ATL can be done without
invasive procedures while keeping a high performance.