Presented by

Dr. Jorge Arévalo Zelada

Laboratorio de Biología Molecular y Celular de TripanosomátidosIMTAvH-UPCH

THE LEISHMANIASES REPRESENT AMAJOR HEALTH PROBLEM IN PERU

AND THE AMERICAS

Incidence in Peru: 32.4/100,000 (1996)

With the exception of Chile, all countries havereported cases of Leishmaniases.

All the clinical forms of Leishmaniasis arepresent in the Americas, including visceralLeishmaniasis.

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

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

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 EBSEEBSEEBS 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

N C S

L.(V.) peruvianaL.(V.) braziliensis

15.8 kb

10.9 kb9.8 kb

3.7 kb

3 kb

Gp63- RFLP analysis

= amplification product 1.3kb= gp63 probe (pLb134-Sp)

coding region 1.8 kb untranslated region 1.3 kb

3 kb gp63 repetition unit

primers

most variable region

TDM1 TDM2

The gp63 unit amplification :

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%

SalI ApaLI ApaI

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

SINTESIS RNA LC 2177

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SINTESIS RNA LCA 01

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DEGRADACION RNA LC 2177

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Cla I Cla I Cla IXhoIXhoI

XhoI XhoIXhoI XhoIEcoRI EcoRI

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

az41 CDEGYSGGLQGFGSSETCAFFRCWGTRWAISLSEQELVS 171az13 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 L AmastigotesML

M= MetacyclicsL=Logaritmic

Clones obtained by Differential Dislay Analysis

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

infection

D a ys p os t in fe c tion

Lesi

on

dia

met

er

(mm

)

10 15 20 25 30 35 40 45 50 55 600 .00

0 .25

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A + (C on tro l)

A + (In fe c te d )

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F ig u re 2 . C o u rse o f in fe c tio n in B A L B /c m ice in o cu la te d w ith 1 0 6 p ro m a s tig o te s o fL . m e xica n a M 3 7 9 . A + : v ita m in A -su ffic ien t d ie t; A -: v ita m in A -d e fic ien t d ie t.

Leuk

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A - (C o n tro l)A - ( In fe c te d )

B e fo rein fe c t io n

6 0 d a y sp o s t in fe c t io n

F ig u re 7 . E f fe c t o f v i ta m in A s ta tu s o n th e to ta l n u m b e r o f le u k o c y te s in p e r ip h e ra lb lo o d f r o m B A L B /c m ic e . A + : v i ta m in A -s u f f ic ie n t d ie t ; A -: v i ta m in A -d e f ic ie n t d ie t .

In f.

D a y s p o s t in fe c tio n

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phoc

ytes

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-2 0 -1 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0In f.0

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A + ( In fe c te d )

A - (C o n tro l)

A - ( In fe c te d )

F ig u re 9 . E ffe c t o f v ita m in A s ta tu s o n th e p e rc e n ta g e o f p e r ip h e ra l b lo o dly m p h o c y te s fro m B A L B /c m ic e . A + : v ita m in A -s u ffic ie n t d ie t; A -: v ita m in A -d e fic ie n td ie t. In f: In fe c tio n .

I n f .

D a y s p o s t in f e c t io n

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trop

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(%

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F i g u r e 1 0 . E f f e c t o f v i t a m i n A s t a t u s o n t h e p e r c e n t a g e o f p e r i p h e r a l b l o o dn e u t r o p h i l s f r o m B A L B / c m i c e . A + : v i t a m i n A - s u f f i c i e n t d i e t ; A - : v i t a m i n A - d e f i c i e n td i e t . I n f : I n f e c t i o n ..

Respuesta Oxidativa de Macrófagos Peritoneales Deficientes de Vitamina A e infectados con

Leishmania mexicana

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control deficienteDieta de Vitamina A

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ac

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Control

Infectado

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