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: 75.042
Session: Diagnostics
Date: Friday, March 12, 2010
Time: 12:30-13:30
Room: Poster & Exhibition Area/Ground Level
Type: Poster Presentation
A highly sensitive reverse transcription polymerase chain reaction method for yellow fever virus
detection
J. Méndez 1 , C. Mendez 2 , C. Domingo 3 , G. Rey 2 , A. Tenorio 4
1 Instituto Nacional de Salud, 01, Bogotá, Colombia, 2 Instituto Nacional de Salud, Bogotá,
Colombia, 3 Robert Koch Institute, Berlin, Germany, 4 Instituto de Salud Carlos III, Madrid, Spain
Background: Diagnosis of YF is an important issue particularly in countries where it is endemic.
Usual serological tests include IgM antibody capture by enzyme-linked immunosorbent assay
(MAC-ELISA), hemagglutination inhibition (HI), complement fixation (CF) and neutralization (N).
Nevertheless, antibody detection is feasible only 5 to 6 days after the offset of symptoms and the
true usefulness is by evaluation of paired samples which are not easy to obtain. On the other
hand, specific diagnosis during infection period is currently done by isolation of virus in mosquito
cells or by genome detection through PCR based methods, while fatal cases are confirmed with
immunohistochemistry with monoclonal antibodies over well conserved fixed tissues. Although
viral isolation is a very specific technique, it takes at last 7 –10 days and sensitivity decrease
when few virus particles are present in the sample or when toxic biliary salts present in serum
inhibit cell growth. Detection of virus in the acute phase must be specific and sensible enough to
confirm the diagnosis, and then start the surveillance measurements.
Methods: Twenty five human sera were processed with the QIAamp Viral RNA Minikit. Reverse
Transcription (RT) was carried out following by Polymerase Chain Reaction (PCR) using specific
primers designed to amplify 734bp from the prM/E junction. Negative samples were reamplified
with internal primers (nested PCR) to obtain a 692bp fragment.
Results: The expected amplification product size was obtained in 20 of 25 samples analyzed.
Positive reaction was obtained in 2 samples after first reaction, while the remaining 18 were
positive after performing nested PCR.
Conclusion: This improved RT-PCR using primers designed in a conserved region of genome to
amplify short fragments, not just ensure detection of all YFV genotypes, but also increase
sensibility even in more difficult samples. Remarkable, this reaction allows rapid differential
diagnosis between Dengue and Yellow Fever, which is absolutely necessary for an effective
surveillance and opportune epidemiologic measures.