**9. References**

266 Current Topics in Tropical Medicine

appropriate" since this is the stage responsible for mammalian disease. In addition, an intracellular *in vitro* model resembles the natural event when the parasite is in the mammalian host. Axenic amastigotes are also employed, although a more efficient

The effectiveness of compounds to kill the parasites has been evaluated using different methodological approaches. Several years ago, direct parasite counting was the most used method (Gaspar., et al. 1992; Chan-Bacab., et al. 2003; Khan., et al. 2003). However, this method lacked accuracy and precision, likely due to human errors. This made necessary to develop new automated methods based on colorimetric, radioactive, fluorescent and luminometer detection (Fumarola., et al. 2004). Colorimetric methods like MTT [3-(4,5 dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] have been used frequently. Recently, these methods are being replaced by transgenic parasites with reporter genes that do not interfere with cellular mitochondria. Parasites genetically engineered to express green fluorescent protein (GFP) or luciferase, have been developed and are currently used in

On the other hand, it is important to measure cytotoxicity to evaluate the possibility that a compound might produce side effects in humans. Mammalian or mouse cell lines are usually employed in these assays. The most used cells are U-937 human histiocytes, TPH-1 human peripheral blood monocytes, and hamster peritoneal macrophages (Robledo., et al. 1999; Weniger., et al. 2001; Varela., et al. 2009; Taylor., et al. 2010). To increase the selectivity of promising drugs, liposomal formulations of the compounds may be evaluated in order to reduce the toxicity as was observed for amphotericin B (Mehta., et

The leishmanicidal activity of compounds that show high anti-leishmanial activity and low toxicity for mammalian cells *in vitro*, is next evaluated *in vivo*. This is normally performed in mouse or the golden hamster (*Mesocricetus auratus*) models depending on the *Leishmania* subgenus (Travi., et al. 2002). Monkey models can also used but these studies are limited to

Through the current chapter, the more relevant techniques for finding drugs and targets employing computational approaches were described. Special considerations should be made when using the homology searching approach for finding drug targets as the context of protein interactions is important for the definition of essentiality. Protein interaction network analysis together with metabolic flux balance analysis are becoming useful alternatives to understand protein function and to systematically select drug targets. The selection of new inhibitory compounds can be done by using virtual screening. Predicted structures can also be used in virtual screening when experimentally derived structures are absent. Finally, machine learning techniques are a promising option to search for antileishmanial drugs, especially when experimental or predicted structures are not available, as may occur with many *Leishmania* proteins. It is important to state that any computational analysis is considered exploratory, and experimental validation is necessary to guide final decisions about potential compounds that can advance to the next stage of drug discovery. However, by using computational tools the search space for drugs and targets is reduced, allowing more focused experiments that could reduce the

alternative is the use of intracellular fluorescent or bioluminescent amastigotes.

automatized protocols involving flow cytometer or luminometer.

only a few laboratories worldwide (Grimaldi., et al. 2010).

al. 1985; Lopez-Berestein 1987).

cost and time of drug development.

**7. Conclusion** 


Current Advances in Computational Strategies for Drug Discovery in Leishmaniasis 269

Giaever, G., A. M. Chu, L. Ni, C. Connelly, L. Riles, S. Veronneau, S. Dow, A. Lucau-Danila,

Glasser, J. S. & C. K. Murray (2011). "Central nervous system toxicity associated with

Good, M. C., J. G. Zalatan & W. A. Lim (2011). "Scaffold proteins: hubs for controlling the

Goodsell, D. S., G. M. Morris & A. J. Olson (1996). "Automated docking of flexible ligands:

Goyeneche-Patino, D. A., L. Valderrama, J. Walker & N. G. Saravia (2008). "Antimony

He, F., Y. Zhang, H. Chen, Z. Zhang & Y. L. Peng (2008). "The prediction of protein-protein

Hermjakob, H., L. Montecchi-Palazzi, C. Lewington, S. Mudali, S. Kerrien, S. Orchard, M.

drug target prioritization in Aspergillus fumigatus." *PLoS Pathog* 3(3): e24. Hubbard, T. J., A. G. Murzin, S. E. Brenner & C. Chothia (1997). "SCOP: a structural

Irwin, J. J. & B. K. Shoichet (2005). "ZINC--a free database of commercially available

Ivens, A. C., C. S. Peacock, E. A. Worthey, L. Murphy, G. Aggarwal, M. Berriman, E. Sisk, M. A.

classification of proteins database." *Nucleic Acids Res* 25(1): 236-239.

compounds for virtual screening." *J Chem Inf Model* 45(1): 177-182.

Leishmania panamensis." *Antimicrob Agents Chemother* 52(12): 4503-4506. Grimaldi, G., Jr., R. Porrozzi, K. Friedrich, A. Teva, R. S. Marchevsky, F. Vieira, N. Miekeley

(Macaca mulatta)." *Antimicrob Agents Chemother* 54(1): 502-505.

interaction networks in rice blast fungus." *BMC Genomics* 9: 519.

liposomal amphotericin B therapy for cutaneous leishmaniasis." *Am J Trop Med Hyg*

resistance and trypanothione in experimentally selected and clinical strains of

& F. J. Paumgartten (2010). "Comparative efficacies of two antimony regimens to treat Leishmania braziliensis-induced cutaneous Leishmaniasis in rhesus macaques

Vingron, B. Roechert, P. Roepstorff, A. Valencia, H. Margalit, J. Armstrong, A. Bairoch, G. Cesareni, D. Sherman & R. Apweiler (2004). "IntAct: an open source molecular interaction database." *Nucleic Acids Res* 32(Database issue): D452-455. Hu, W., S. Sillaots, S. Lemieux, J. Davison, S. Kauffman, A. Breton, A. Linteau, C. Xin, J.

Bowman, J. Becker, B. Jiang & T. Roemer (2007). "Essential gene identification and

Rajandream, E. Adlem, R. Aert, A. Anupama, Z. Apostolou, P. Attipoe, N. Bason, C. Bauser, A. Beck, S. M. Beverley, G. Bianchettin, K. Borzym, G. Bothe, C. V. Bruschi, M. Collins, E. Cadag, L. Ciarloni, C. Clayton, R. M. Coulson, A. Cronin, A. K. Cruz, R. M. Davies, J. De Gaudenzi, D. E. Dobson, A. Duesterhoeft, G. Fazelina, N. Fosker, A. C. Frasch, A. Fraser, M. Fuchs, C. Gabel, A. Goble, A. Goffeau, D. Harris, C. Hertz-Fowler, H. Hilbert, D. Horn, Y. Huang, S. Klages, A. Knights, M. Kube, N. Larke, L. Litvin, A.

Saccharomyces cerevisiae genome." *Nature* 418(6896): 387-391.

flow of cellular information." *Science* 332(6030): 680-686.

applications of AutoDock." *J Mol Recognit* 9(1): 1-5.

86(1): 41-49.

84(4): 566-568.

nanoparticles against intracellular Leishmania donovani." *Ann Trop Med Parasitol*

K. Anderson, B. Andre, A. P. Arkin, A. Astromoff, M. El-Bakkoury, R. Bangham, R. Benito, S. Brachat, S. Campanaro, M. Curtiss, K. Davis, A. Deutschbauer, K. D. Entian, P. Flaherty, F. Foury, D. J. Garfinkel, M. Gerstein, D. Gotte, U. Guldener, J. H. Hegemann, S. Hempel, Z. Herman, D. F. Jaramillo, D. E. Kelly, S. L. Kelly, P. Kotter, D. LaBonte, D. C. Lamb, N. Lan, H. Liang, H. Liao, L. Liu, C. Luo, M. Lussier, R. Mao, P. Menard, S. L. Ooi, J. L. Revuelta, C. J. Roberts, M. Rose, P. Ross-Macdonald, B. Scherens, G. Schimmack, B. Shafer, D. D. Shoemaker, S. Sookhai-Mahadeo, R. K. Storms, J. N. Strathern, G. Valle, M. Voet, G. Volckaert, C. Y. Wang, T. R. Ward, J. Wilhelmy, E. A. Winzeler, Y. Yang, G. Yen, E. Youngman, K. Yu, H. Bussey, J. D. Boeke, M. Snyder, P. Philippsen, R. W. Davis & M. Johnston (2002). "Functional profiling of the


Chang, M. W., C. Ayeni, S. Breuer & B. E. Torbett (2010). "Virtual screening for HIV protease inhibitors: a comparison of AutoDock 4 and Vina." *PLoS One* 5(8): e11955. Chavali, A. K., J. D. Whittemore, J. A. Eddy, K. T. Williams & J. A. Papin (2008). "Systems

Chua, H. N., W. K. Sung & L. Wong (2006). "Exploiting indirect neighbours and topological

Das, B. B., A. Ganguly & H. K. Majumder (2008). "DNA topoisomerases of Leishmania: the potential targets for anti-leishmanial therapy." *Adv Exp Med Biol* 625: 103-115. David, H., M. Akesson & J. Nielsen (2003). "Reconstruction of the central carbon metabolism

de Azevedo, W. F., Jr. & M. B. Soares (2009). "Selection of targets for drug development

Dreze, M., D. Monachello, C. Lurin, M. E. Cusick, D. E. Hill, M. Vidal & P. Braun (2010). "High-quality binary interactome mapping." *Methods Enzymol* 470: 281-315. Duarte, N. C., M. J. Herrgard & B. O. Palsson (2004). "Reconstruction and validation of

Eberle, C., B. S. Lauber, D. Fankhauser, M. Kaiser, R. Brun, R. L. Krauth-Siegel & F.

Enright, A. J., S. Van Dongen & C. A. Ouzounis (2002). "An efficient algorithm for large-scale

Faraut-Gambarelli, F., R. Piarroux, M. Deniau, B. Giusiano, P. Marty, G. Michel, B. Faugere

Finn, R. D., M. Marshall & A. Bateman (2005). "iPfam: visualization of protein-protein

Finn, R. D., J. Mistry, B. Schuster-Bockler, S. Griffiths-Jones, V. Hollich, T. Lassmann, S.

Florez, A. F., D. Park, J. Bhak, B. C. Kim, A. Kuchinsky, J. H. Morris, J. Espinosa & C.

major as a tool for drug target selection." *BMC Bioinformatics* 11: 484. Forsyth, R. A., R. J. Haselbeck, K. L. Ohlsen, R. T. Yamamoto, H. Xu, J. D. Trawick, D. Wall, L.

essential genes in Staphylococcus aureus." *Mol Microbiol* 43(6): 1387-1400. Fumarola, L., R. Spinelli & O. Brandonisio (2004). "In vitro assays for evaluation of drug

Gaspar, R., F. R. Opperdoes, V. Preat & M. Roland (1992). "Drug targeting with

activity against Leishmania spp." *Res Microbiol* 155(4): 224-230.

detection of protein families." *Nucleic Acids Res* 30(7): 1575-1584.

leishmaniasis." *Antimicrob Agents Chemother* 41(4): 827-830.

Saccharomyces cerevisiae iND750, a fully compartmentalized genome-scale

Diederich (2011). "Improved inhibitors of trypanothione reductase by combination of motifs: synthesis, inhibitory potency, binding mode, and antiprotozoal

& H. Dumon (1997). "In vitro and in vivo resistance of Leishmania infantum to meglumine antimoniate: a study of 37 strains collected from patients with visceral

interactions in PDB at domain and amino acid resolutions." *Bioinformatics* 21(3):

Moxon, M. Marshall, A. Khanna, R. Durbin, S. R. Eddy, E. L. Sonnhammer & A. Bateman (2006). "Pfam: clans, web tools and services." *Nucleic Acids Res* 34(Database

Muskus (2010). "Protein network prediction and topological analysis in Leishmania

Wang, V. Brown-Driver, J. M. Froelich, K. G. C, P. King, M. McCarthy, C. Malone, B. Misiner, D. Robbins, Z. Tan, Z. Y. Zhu Zy, G. Carr, D. A. Mosca, C. Zamudio, J. G. Foulkes & J. W. Zyskind (2002). "A genome-wide strategy for the identification of

polyalkylcyanoacrylate nanoparticles: in vitro activity of primaquine-loaded

of Aspergillus niger." *Eur J Biochem* 270(21): 4243-4253.

metabolic model." *Genome Res* 14(7): 1298-1309.

activities." *ChemMedChem* 6(2): 292-301.

against protozoan parasites." *Curr Drug Targets* 10(3): 193-201.

*Syst Biol* 4: 177.

22(13): 1623-1630.

410-412.

issue): D247-251.

analysis of metabolism in the pathogenic trypanosomatid Leishmania major." *Mol* 

weight to predict protein function from protein-protein interactions." *Bioinformatics*

nanoparticles against intracellular Leishmania donovani." *Ann Trop Med Parasitol* 86(1): 41-49.


Current Advances in Computational Strategies for Drug Discovery in Leishmaniasis 271

Oliveira, L. F., A. O. Schubach, M. M. Martins, S. L. Passos, R. V. Oliveira, M. C. Marzochi &

Park, D., S. Lee, D. Bolser, M. Schroeder, M. Lappe, D. Oh & J. Bhak (2005). "Comparative

Leishmania species that cause diverse human disease." *Nat Genet* 39(7): 839-847. Perez-Victoria, J. M., A. Di Pietro, D. Barron, A. G. Ravelo, S. Castanys & F. Gamarro (2002).

in Leishmania: a search for reversal agents." *Curr Drug Targets* 3(4): 311-333. Peri, S., J. D. Navarro, T. Z. Kristiansen, R. Amanchy, V. Surendranath, B. Muthusamy, T. K.

Pieper, U., N. Eswar, B. M. Webb, D. Eramian, L. Kelly, D. T. Barkan, H. Carter, P. Mankoo,

Plata, G., T. L. Hsiao, K. L. Olszewski, M. Llinas & D. Vitkup (2010). "Reconstruction and flux-

Reed, J. L. & B. O. Palsson (2003). "Thirteen years of building constraint-based in silico

Rives, A. W. & T. Galitski (2003). "Modular organization of cellular networks." *Proc Natl* 

Robledo, S. M., A. Z. Valencia & N. G. Saravia (1999). "Sensitivity to Glucantime of Leishmania viannia isolated from patients prior to treatment." *J Parasitol* 85(2): 360-366. Sakharkar, K. R., M. K. Sakharkar & V. T. Chow (2004). "A novel genomics approach for the

Shannon, P., A. Markiel, O. Ozier, N. S. Baliga, J. T. Wang, D. Ramage, N. Amin, B.

resources." *Nucleic Acids Res* 37(Database issue): D347-354.

models of Escherichia coli." *J Bacteriol* 185(9): 2692-2699.

(protein structural interactome map)." *Bioinformatics* 21(15): 3234-3240. Peacock, C. S., K. Seeger, D. Harris, L. Murphy, J. C. Ruiz, M. A. Quail, N. Peters, E. Adlem,

leishmaniasis treatment in the New World." *Acta Trop* 118(2): 87-96. Osman, A. (2004). "Yeast two-hybrid assay for studying protein-protein interactions."

*Antimicrob Agents Chemother* 28(4): 511-513.

*Methods Mol Biol* 270: 403-422.

32(Database issue): D497-501.

*Acad Sci U S A* 100(3): 1128-1133.

aeruginosa." *In Silico Biol* 4(3): 355-360.

amphotericin B and a muramyl dipeptide analog, alone and in combination."

C. A. Andrade (2011). "Systematic review of the adverse effects of cutaneous

interactomics analysis of protein family interaction networks using PSIMAP

A. Tivey, M. Aslett, A. Kerhornou, A. Ivens, A. Fraser, M. A. Rajandream, T. Carver, H. Norbertczak, T. Chillingworth, Z. Hance, K. Jagels, S. Moule, D. Ormond, S. Rutter, R. Squares, S. Whitehead, E. Rabbinowitsch, C. Arrowsmith, B. White, S. Thurston, F. Bringaud, S. L. Baldauf, A. Faulconbridge, D. Jeffares, D. P. Depledge, S. O. Oyola, J. D. Hilley, L. O. Brito, L. R. Tosi, B. Barrell, A. K. Cruz, J. C. Mottram, D. F. Smith & M. Berriman (2007). "Comparative genomic analysis of three

"Multidrug resistance phenotype mediated by the P-glycoprotein-like transporter

Gandhi, K. N. Chandrika, N. Deshpande, S. Suresh, B. P. Rashmi, K. Shanker, N. Padma, V. Niranjan, H. C. Harsha, N. Talreja, B. M. Vrushabendra, M. A. Ramya, A. J. Yatish, M. Joy, H. N. Shivashankar, M. P. Kavitha, M. Menezes, D. R. Choudhury, N. Ghosh, R. Saravana, S. Chandran, S. Mohan, C. K. Jonnalagadda, C. K. Prasad, C. Kumar-Sinha, K. S. Deshpande & A. Pandey (2004). "Human protein reference database as a discovery resource for proteomics." *Nucleic Acids Res*

R. Karchin, M. A. Marti-Renom, F. P. Davis & A. Sali (2009). "MODBASE, a database of annotated comparative protein structure models and associated

balance analysis of the Plasmodium falciparum metabolic network." *Mol Syst Biol* 6: 408.

identification of drug targets in pathogens, with special reference to Pseudomonas

Schwikowski & T. Ideker (2003). "Cytoscape: a software environment for integrated models of biomolecular interaction networks." *Genome Res* 13(11): 2498-2504.

Lord, T. Louie, M. Marra, D. Masuy, K. Matthews, S. Michaeli, J. C. Mottram, S. Muller-Auer, H. Munden, S. Nelson, H. Norbertczak, K. Oliver, S. O'Neil, M. Pentony, T. M. Pohl, C. Price, B. Purnelle, M. A. Quail, E. Rabbinowitsch, R. Reinhardt, M. Rieger, J. Rinta, J. Robben, L. Robertson, J. C. Ruiz, S. Rutter, D. Saunders, M. Schafer, J. Schein, D. C. Schwartz, K. Seeger, A. Seyler, S. Sharp, H. Shin, D. Sivam, R. Squares, S. Squares, V. Tosato, C. Vogt, G. Volckaert, R. Wambutt, T. Warren, H. Wedler, J. Woodward, S. Zhou, W. Zimmermann, D. F. Smith, J. M. Blackwell, K. D. Stuart, B. Barrell & P. J. Myler (2005). "The genome of the kinetoplastid parasite, Leishmania major." *Science* 309(5733): 436-442.


Jeong, H., S. P. Mason, A. L. Barabasi & Z. N. Oltvai (2001). "Lethality and centrality in

Kamath, R. S., A. G. Fraser, Y. Dong, G. Poulin, R. Durbin, M. Gotta, A. Kanapin, N. Le Bot,

Khan, K. M., M. Rasheed, U. Zia, S. Hayat, F. Kaukab, M. I. Choudhary, R. Atta ur & S.

Kim, J. G., D. Park, B. C. Kim, S. W. Cho, Y. T. Kim, Y. J. Park, H. J. Cho, H. Park, K. B. Kim,

Klipp, E., Herwig, R., Kowald, A.,Wierling, C. & Lehrach, H (2005). *Systems Biology in* 

Knox, C., V. Law, T. Jewison, P. Liu, S. Ly, A. Frolkis, A. Pon, K. Banco, C. Mak, V. Neveu,

Kuhn, M., D. Szklarczyk, A. Franceschini, M. Campillos, C. von Mering, L. J. Jensen, A.

Lin, C. Y., C. H. Chin, H. H. Wu, S. H. Chen, C. W. Ho & M. T. Ko (2008). "Hubba: hub

Lopez-Berestein, G. (1987). "Liposomes as carriers of antimicrobial agents." *Antimicrob* 

Maltezou, H. C. (2008). "Visceral leishmaniasis: advances in treatment." *Recent Pat Antiinfect* 

McInnes, C. (2007). "Virtual screening strategies in drug discovery." *Curr Opin Chem Biol*

Mehta, R. T., G. Lopez-Berestein, R. L. Hopfer, K. Mehta, R. A. White & R. L. Juliano (1985).

molecules and proteins." *Nucleic Acids Res* 38(Database issue): D552-556. Lang, T., S. Goyard, M. Lebastard & G. Milon (2005). "Bioluminescent Leishmania

parasitism features in living mice." *Cell Microbiol* 7(3): 383-392.

biology." *Nucleic Acids Res* 36(Web Server issue): W438-443.

S. Moreno, M. Sohrmann, D. P. Welchman, P. Zipperlen & J. Ahringer (2003). "Systematic functional analysis of the Caenorhabditis elegans genome using RNAi."

Perveen (2003). "Synthesis and in vitro leishmanicidal activity of some hydrazides

K. O. Yoon, S. J. Park, B. M. Lee & J. Bhak (2008). "Predicting the interactome of Xanthomonas oryzae pathovar oryzae for target selection and DB service." *BMC* 

*Practice: Concepts, Implementation and Application.* Wiley-VCH Verlag GmbH & Co.

Y. Djoumbou, R. Eisner, A. C. Guo & D. S. Wishart (2011). "DrugBank 3.0: a comprehensive resource for 'omics' research on drugs." *Nucleic Acids Res*

Beyer & P. Bork (2010). "STITCH 2: an interaction network database for small

expressing luciferase for rapid and high throughput screening of drugs acting on amastigote-harbouring macrophages and for quantitative real-time monitoring of

objects analyzer--a framework of interactome hubs identification for network

"Prophylaxis of murine candidiasis via application of liposome-encapsulated

309(5733): 436-442.

*Nature* 421(6920): 231-237.

*Bioinformatics* 9: 41.

39(Database issue): D1035-1041.

*Agents Chemother* 31(5): 675-678.

*Drug Discov* 3(3): 192-198.

11(5): 494-502.

protein networks." *Nature* 411(6833): 41-42.

and their analogues." *Bioorg Med Chem* 11(7): 1381-1387.

KGaA, ISBN: 9783527310784, Weinheim, Germany.

Lord, T. Louie, M. Marra, D. Masuy, K. Matthews, S. Michaeli, J. C. Mottram, S. Muller-Auer, H. Munden, S. Nelson, H. Norbertczak, K. Oliver, S. O'Neil, M. Pentony, T. M. Pohl, C. Price, B. Purnelle, M. A. Quail, E. Rabbinowitsch, R. Reinhardt, M. Rieger, J. Rinta, J. Robben, L. Robertson, J. C. Ruiz, S. Rutter, D. Saunders, M. Schafer, J. Schein, D. C. Schwartz, K. Seeger, A. Seyler, S. Sharp, H. Shin, D. Sivam, R. Squares, S. Squares, V. Tosato, C. Vogt, G. Volckaert, R. Wambutt, T. Warren, H. Wedler, J. Woodward, S. Zhou, W. Zimmermann, D. F. Smith, J. M. Blackwell, K. D. Stuart, B. Barrell & P. J. Myler (2005). "The genome of the kinetoplastid parasite, Leishmania major." *Science*

amphotericin B and a muramyl dipeptide analog, alone and in combination." *Antimicrob Agents Chemother* 28(4): 511-513.


**17** 

*Argentina* 

**Advances in Serological Diagnosis of Chagas'** 

**Disease by Using Recombinant Proteins** 

*1Laboratorio de Tecnología Inmunológica (LTI), Fac. Bioquímica y Cs. Biológicas,* 

*2Instituto de Química Rosario (IQUIR-CONICET), División Química Analítica,* 

Chagas' disease is an infection caused by the parasite *Trypanosoma cruzi*, mainly occurring in American countries where the parasite vector bug, *Triatoma infestans*, is widespread. One hundred million individuals are currently under threaten of infection, as well as 16 million people are considered affected by the illness in Latin America alone.(Editorial, 2009) Considering indicators such as the disability-adjusted life years, DALY, and from a social point of view, Chagas' disease accounts for the third most important tropical illness of the World, following malaria and schistosomiasis.(Bitran *et al.*, 2009) Moreover, Chagas disease epidemiology nowadays impacts in non-endemic regions due to globalization, being the infection disseminated all over the world (Gascon *et al.* 2009). Certainly, the non-vectorial disease transmission (mother to child, transfusional and by organ transplantation) is the way the illness spreads in Europe, North America, Japan, and Australia, because many infected people have migrated from endemic regions to distant cities. (Bowling & Walter,

The parasite distribution and living habits of rural Latin-American people have determined that the main transmission route is the vectorial one (via *Triatoma infestans*), leading to up 80% of human infection.(WHO, 2003) The main contagion way in urban areas arise from blood-transfusion, being responsible for 5-20% of the reported cases, while vertical transmission (mother-to-child) accounts for 2-10% of the infections.(Carlier

The infection transmission by oral route because of consumption of contaminated food and drinks is lower than that reported for the previously-mentioned routes.(Dias *et al.*, 2011) However, it has to be taken into account that the success of the non-oral infection prevention has increased the importance of the oral route of transmission.(Dias *et al.*, 2011; Nóbrega *et al.*, 2009) It is noteworthy that the acute outcome of the oral infection is particularly

From the clinical point of view, the illness presents variable unspecific symptoms, depending on its stage: the acute one, shortly after primary infection, and the chronic stage, which may last many years if the individual is not treated. Human infection typically

**1. Introduction** 

& Torrico, 2003)

severe.(Bastos et al 2010).

2009; Lescure *et al.*, 2009; Yadon & Schmunis, 2009)

*Fac. Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario* 

Iván S. Marcipar1 and Claudia M. Lagier2

*Universidad Nacional del Litoral* 

