This document presents a study on genetic markers for drug resistance in Leishmania parasites. The study aimed to characterize clinical isolates of Leishmania donovani from India in terms of their phenotypes, genotypes, and any relationship to drug resistance. The methods section describes culturing reference and clinical Leishmania strains, isolating genomic DNA, using PCR-RFLP and SSCP to detect polymorphisms, and susceptibility assays to test sensitivity to sodium stibogluconate (SSG). The results section finds polymorphisms in the ITS1, ITS2, hsp70 and anonymous DNA fragments that differentiate SSG-resistant from sensitive parasites. The discussion reviews how SSCP can identify single nucleotide changes and how understanding genetic

1. Presented by: - DAVID GAVIRIA ORTIZ
-ANA MARÍA GÓMEZ GÓMEZ

2. BODY OF OUR PRESENTATION
INTRODUCTION.
AIM OF THE STUDY.
METHODS.
RESULTS.
DISCUSSION.
CLOSURES.
CONCEPT MAPS.

3. INTRODUCTION

4. GENETIC
MARKERS
LEISHMANIASIS
DRUG
RESISTANCE

5. • Female phlebotomine
sandflies
• Promastigote
• Amastigote

6. L I F E
C Y C L E

7. GENERAL
OBJECTIVE
Characterize each clinical isolate of
Indian KA phenotypes
genotypes
attempting to the development of
markers for drug resistance, if any.

8. MATERIALES Y MÉTODOS
DECLARACIONES
ÉTICAS

9. CULTIVO DE PARÁSITOS DE
LEISHMANIA Y CEPAS DE
REFERENCIA
Cepas de referencia de
- L. donovani (DD8)
- L. tropica (K27)
- L. donovani (MHOM/IN/1983/AG83)

10. CEPAS CONTROL

11. Amastigotes
confirmados con
Giemsa.
https://www.cdc.gov/dpdx/leishmaniasis/

12. AISLAMIENTO DE
DNA GENÓMICO

13. PCR-RFLP
• Polimorfismos.
• Enzimas de restricción.
HaeIII

14. MURINE
MACROPHAGE
LIKE TUMOR
CELL

15. ENSAYO DE
SUSCEPTIBILIDAD
• Para ver si hay resistencia o sensibilidad
a una droga.

16. L. donovani
SSG
Macrófagos
Promastigotes

17. SSCP
• Detectar mutaciones y genotipar.
• 4 pasos.
Fragmentos de
control
• L0110
• L510
• L413
• LK413
• L0308
• L0114
Fragmentos usados
• ITS1
• ITS2

18. RESULTADOS

19. SSG
Estibogluconato de sodio
SSCP
Polimorfismo conformacional de hebra sencilla

20. ITS1

21. hsp70

22. S
S
R
R
AI: >3.0 R
<3.0 S

23. ITS1 A
B
C
D
L. donovani
L. tropica
SSG-R
ITS2 X
Y
Z
L. donovani
L. tropica SSG-R

25. Fragmentos anónimos de DNA
PCR
especies SSG R – S
Q
R
S
≠ Patrones polimórficos
SSG R

27. DISCUSSION

28. AUTHOR WHAT THEY SAID OUTCOME
SSCP is one of the basic methods to
identify single base divergence in genes.Gasser
Understanding the molecular
mechanisms of clinical resistance is
crucial for analyse the progress and
extent of drug resistance in the field.
Asutosh
Srivastava
Significant genetic polymorphisms exist
in L. donovani strains in Bihar, India.
You can differenciate resistant and
sensitive SSG Leishmania parasites,
by protein encoding genes, rDNA and
mini-exon.
Downing

29. CLOSURES
• For this pathology, it isn’t enough for the sandflies to transmit the parasites
just by a bite, it is obligatory an intra-macrophage process.
• One can tell apart the resistant Leishmania isolates from the sensitive
Leishmania isolates on the SSCP method.

30. • In order to avoid drug resistance, we need to understand the
polymorphisms regarding each parasite.
• Of the few samples that were positive for the codification of L. tropica,
they could recognize the susceptibility for the SSG. Discovering an
important, but not of huge magnitude diagnosis impression.

31. ANA’S CONCEPT MAP

32. DAVID´S CONCEPT MAP

33. Thank you!