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Presented by Dr. Jorge Arévalo Zelada Laboratorio de Biología Molecular y Celular de Tripanosomátidos IMTAvH-UPCH

Presented by Dr. Jorge Arévalo Zelada Laboratorio de Biología Molecular y Celular de Tripanosomátidos IMTAvH-UPCH. GENERAL AIM OF THE LEISHMANIASIS RESEARCH GROUP

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Presented by Dr. Jorge Arévalo Zelada Laboratorio de Biología Molecular y Celular de Tripanosomátidos IMTAvH-UPCH

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  1. Presented by Dr. Jorge Arévalo Zelada Laboratorio de Biología Molecular y Celular de Tripanosomátidos IMTAvH-UPCH

  2. GENERAL AIM OF THE LEISHMANIASIS RESEARCH GROUP • To understand parasite and environment contributions to disease transmission and disease evolution using molecular tools to track down phenotype polymorphism and parasite population heterogeneity. These features might be associated to disease manifestations. • To develop molecular tools for better diagnostic procedures, specially under conditions where they show clear cost/benefit comparative advantages over conventional laboratory diagnosis.

  3. SPECIFIC OBJECTIVES WITHIN DGIS FRAMEWORK • Formal assessment of diagnostic performance of Leishmania DNA detection procedures within a hospital service attending skin ulcers. • Development of new DNA targets and methodologies to improve parasite detection and to be capable of Leishmania identification in skin biopsies without the need of culture. • To transfer know how to other research groups at the IMTAvH. • To develop human resources with skills and material facilities to carry out gene expression studies. • To develop an animal model that allows to study the possible links between Vitamin A, immunological response and disease course infection.

  4. Diagnostic performance of Leishmania DNA detection procedures within a hospital service • · Joint work with the Clinical component through the skin ulcer project. • · Laboratory responsible: Leyda Cabrera • · Clinical responsible: Tine Verdonck

  5. 1 2 3 4 5 6 7 8 9 10 11 12 13 14

  6. Patients attending the IMTAvH • Samples provided by the Clinical component • DNA extraction and PCR detection procedures • Hybridization and non radioactive detection procedures • Diagnostic results delivered according health service needs

  7. New targets and methodologies For detection and identification of leishmania in skin biopsies • · Joint work with the Protozoology unit. • · Laboratory responsible: Kathleen Victoir & Jean Claude Dujardin • Clinical responsible: Alejandro LLanos

  8. Gp63 gene organization and DNA sequencing at the IMT (Antwerp) • Former tests with cultures parasites at the IMT (Antwerp) • Assays with biopsies from experimentally infected animals at the IMTAvH (Lima) • Assays with biopsies from patients (Lima and Antwerp)

  9. EEBS EBS B B BE BSE BS G3411 EBS E B B B B B BS EBS EBS EEBSEEBS EBS C4121 BB BB BB BS E SB B BEB BS B B B E BSE B C1211 ** ** E S E S E S S E SBS ES ES ESBS BSS ESS BS SBEBSB C711/D9311 B B B B B B B B B B B B B B S E S E S E S S E S A811 B B B B B B B B B B B B B B B B B B B B B B B9211 5 kb Gp63 gene locus (partial) in L.(V.)braziliensis

  10. Gp63- RFLP analysis 15.8 kb 10.9 kb 9.8 kb 3.7 kb 3 kb N C S L.(V.) braziliensis L.(V.) peruviana

  11. Sensitivity of gp63 PCR detection • 22 samples analysed by kDNA PCR (all +), gp63 PCR and parasitology • gp63 PCR: 91% • parasitology: 27%

  12. Sensitivity and clinical form • 7 cutaneous samples (kDNA+): gp63 PCR: 100% parasitology: 57% • 15 mucosal samples (kDNA+): gp63 PCR: 86% parasitology: 13%

  13. PCR-RFLP (x = biopsy) x x b p g b p l x x x g x b p SalI ApaLI ApaI

  14. Conclusions • Gp63 PCR-RFLP: complementary tool • plus: discrimination of species and intra-specific populations (NW and OW) • applications: prognosis, epidemiological surveillance, imported pathologies...

  15. Prospects • Increasing sensitivity of detection (nested et al.) • Simplification of the assay • Alternative genomic targets • Link with biological features

  16. Thanks • K.Victoir, S.De Doncker, M.Boelaert & D.Le Ray: ITG Antwerpen • L.Cabrera, E.Alvarez, A.Llanos-Cuentas & J.Arevalo: IMTAvH Lima • S.Rijal: BPKIHS, Dharan, Nepal • S.Guerbouj, IPT Tunis • N.Nuwayri-Salti, AUB Beyruth EC, DGIS, FWO

  17. OTHER TARGETS • The following are not sequences developed with DGIS direct support but they will be used to design DNA primers that will be used under the same concept of gp63 genes. • · rDNA InteGenic Sequences (IGS) • · Cistein proteinase b (cpb) like genes

  18. IGS Restriction Map of Leishmania peruviana, HB44 strain Promotora ETS Figure 1.. IGS from HB44 was amplified and cloned into TOPO XL. A recombinant clone , N44 containing a 6500pb insert. The Figure also shows the place of primers annealing sites (arrows) It also depicts the beta and delta sequences (blue bars underlined)

  19. IGS Restriction Map of Leishmania peruviana, LCA04 strain 2000 Figure 2.. IGS from LCA 04 was amplified and cloned into TOPO XL. A recombinant clone , S04 containing a 5500pb insert. The Figure also shows the place of primers annealing sites (arrows) It also depicts the beta and delta sequences (blue bars underlined)

  20. Cla I XhoI Cla I XhoI Cla I XhoI EcoRI XhoI XhoI EcoRI XhoI 1800 bp 2300 bp 3000 bp PROPOSED MODEL WITH A THREE CP GENES TANDEM ARRAY The 5´end gene (1800 bp) was cloned in AZ41 and the central gene in AZ13

  21. az41CDEGYSGGLQGFGSSETCAFFRCWGTRWAISLSEQELVS 171 az13 TDQGMCGSCWAFSAIGNIESQWYLATHSLISLSEQELVS 180 *:* .*. .*.: . .*: **********

  22. IntramuRal know how transfer • · Joint work with the Mycology unit of the IMTAvH. • · Laboratory responsible: whole unit. • Edgar Neyra, who was member of the Leishmaniasis research unit, onset the setting up of the molecular biology lab at the Mycology unit. In addition, Livia Santibañez has been also recruited recently.

  23. Gene expression studies. • · Joint work with the Protozoology unit. • · Laboratory responsible: Dionicia Gamboa & Jean-Claude Dujardin

  24. Training of Dionicia Gamboa in basic molecular biology techniques (Antwerp) Training of Dionisia Gamboa in Differential Display (Maastrich) Set up Differential Display at Antwerp Initial training on DNA cloning techniques (Lima) Identification of target sequence candidates, cloning and sequencing (Antwerp) Set up Differential Display in Lima Identification of other target sequence candidates and working with other Leishmania isolates (Lima) PhD Thesis

  25. Amastigotes M L ML M= Metacyclics L=Logaritmic

  26. Clones obtained by Differential Dislay Analysis

  27. Animal model for Vitamin A defficiencies, immunological response and disease course infection

  28. THE NEXT STEP • We aim to become a Molecular Epidemiology Unit that will extend our expertise gained with Leishmaniasis to other infectious diseases relevant to the public health of Peru and other countries of Latinamerica. Two approaches will be undertaken: • · Starting new research lines with other pathogens (for example drug resistance) • · Supporting molecular research of other groups at the IMTAvH

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