Dr. Jorge Ar

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

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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
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1 2 3 4 5 6 7 8 9
10 11 12 13 14
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Patients attending the IMTAvH Samples
provided by the Clinical component DNA
extraction and PCR detection procedures Hybridi
zation and non radioactive detection
procedures Diagnostic results delivered
according health service needs
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• 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

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• 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)

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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
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Gp63- RFLP analysis
15.8 kb
10.9 kb
9.8 kb
3.7 kb
3 kb
N
C
S
L.(V.) peruviana
L.(V.) braziliensis
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Sensitivity of gp63 PCR detection

• 22 samples analysed by kDNA PCR (all ), gp63 PCR
  and parasitology
• gp63 PCR 91
• parasitology 27

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Sensitivity and clinical form

• 7 cutaneous samples (kDNA)
• gp63 PCR 100
• parasitology 57
• 15 mucosal samples (kDNA)
• gp63 PCR 86
• parasitology 13

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PCR-RFLP (x biopsy)
x x b p g b p l x x x g
x b p
SalI ApaLI ApaI
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Conclusions

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

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Prospects

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

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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

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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
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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)
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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)
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Cla I
XhoI
Cla I
Cla I
XhoI
XhoI
XhoI
XhoI
XhoI
EcoRI
EcoRI
1800 bp
2300 bp
3000 bp
PROPOSED MODEL WITH A THREE CP GENES TANDEM
ARRAY The 5end gene (1800 bp) was cloned in
AZ41 and the central gene in AZ13
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az41 CDEGYSGGLQGFGSSETCAFFRCWGTRWAISLSEQELVS
171 az13 TDQGMCGSCWAFSAIGNIESQWYLATHSLISLSEQELVS
180 .. .. . .
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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.
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Gene expression studies. Joint work with
the Protozoology unit. Laboratory responsible
Dionicia Gamboa Jean-Claude Dujardin
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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
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M L
Amastigotes
ML
M Metacyclics LLogaritmic
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Clones obtained by Differential Dislay Analysis
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Animal model for Vitamin A defficiencies,
immunological response and disease course
infection
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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