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

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.

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.

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

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

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

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)

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

NCS L.(V.) peruvianaL.(V.) braziliensis 15.8 kb 10.9 kb 9.8 kb 3.7 kb 3 kb Gp63- RFLP analysis

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

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

SalIApaLIApaI x x b p g b p l x x x g x b p PCR-RFLP (x = biopsy)

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

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

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

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

IGS Restriction Map of Leishmania peruviana, HB44 strain 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) Promotora ETS

IGS Restriction Map of Leishmania peruviana, LCA04 strain 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) 2000

Cla I XhoI EcoRI 1800 bp2300 bp3000 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

az41 CDEGYSGGLQGFGSSETCAFFRCWGTRWAISLSEQELVS 171 az13 TDQGMCGSCWAFSAIGNIESQWYLATHSLISLSEQELVS 180 *:*.*..*.:..*: **********

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.

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

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

M LAmastigotes ML M= Metacyclics L=Logaritmic

Clones obtained by Differential Dislay Analysis

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

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