Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes

Cuba Cuba, C.A.; Torno, C.O.; Ledesma, O.; Visciarelli, E.; Garcia, S.; Prat, M.I.; Costamagna, R.; Barbieri, L.; Evans, D.A.

doi:10.1590/S0036-46651996000600005

Abstracts

Diagnostic and parasite characterization and identification studies were carried out in human patients with cutaneous leishmaniasis lesions in Santiago del Estero, Northern Province of Argentina. Diagnostic procedures were biopsies of lesions for smears and inoculations in hamster, needle aspirations of material from ulcers for "in vitro" cultures. Immunodiagnostic techniques applied were IFAT-IgG and Montenegro skin test. Primary isolation of eight stocks of leishmanial parasites was achieved from patients with active lesions. All stocks were biologically characterized by their behaviour in hamster, measurements of amastigote and promastigotes and growth "in vitro". Eight stocks were characterized and identified at species level by their reactivity to a cross-panel of sub-genus and specie-specific Monoclonal Antibodies through an Indirect Immunofluorescence technique and a Dot-ELISA. We conclude from the serodeme analysis of Argentina stocks that: stocks MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 and SE-36 are Leishmania (Viannia) braziliensis. Three Leishmania stocks (SE-1; SE-2 and SE-30) did not react with one highly specie-specific Monoclonal Antibody (Clone: B-18, Leishmania (Viannia) braziliensis marker) disclosing two serodeme group patterns. Five out of eight soluble extracts of leishmanial promastigotes were electrophoresed on thin-layer starch gels and examined for the enzyme MPI, Mannose Phosphate Isomerase; MDH, Malate Dehydrogenase; 6PGD, 6 Phosphogluconate Dehydrogenase; NH, Nucleoside Hydrolase, 2-deoxyinosinc as substrate; SOD, Superoxide Dismutase; GPI, Glucose Phosphate Isomerase and ES, Esterase. From the isoenzyme studies we concluded that stocks: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8 and SE-8-I are isoenzymatically Leishmania (Viannia) braziliensis. We need to analyze more enzymes before assigning them to a braziliensis zymodeme.

Cutaneous leishmaniasis; Leishmania (Viannia) braziliensis; Identification; Santiago del Estero; Argentina

Estudos de diagnóstico, caracterização parasitária e identificação foram conduzidos em pacientes humanos com lesões cutâneas de leishmaniose na Província de Santiago del Estero, no Norte da Argentina. Os procedimentos de diagnóstico foram: biópsias de lesões para utilização em esfregaços e inoculação em hamster; aspiração (com agulha) de úlceras, para cultura "in vitro". As técnicas imunodiagnósticas empregadas foram a IFAT-IgG e o teste intradérmico de Montenegro. Oito cepas de parasitas foram isoladas, sendo estas obtidas de pacientes com lesões ativas. Todas as cepas foram inicialmente caracterizadas biologicamente por seu comportamento na infecção experimental do hamster, mensuração dos amastigotas e promastigotas e crescimento "in vitro". As mesmas oito cepas foram logo identificadas e caracterizadas a nível de espécie, devido a sua reatividade frente a um painel de anticorpos monoclonais subgênero e espécie-específicos. Isso foi realizado utilizando o teste de Imunofluorescência Indireta (IFAT/MAbs) e de um procedimento de Dot-ELISA. Nós concluímos a partir da análise de serodema dos isolados argentinos que: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 e SE-36 são Leishmania (Viannia) braziliensis. Entretanto, três dos isolados de Leishmania (SE-1; SE-2 e SE-30) não foram reconhecidos quando testados com um anticorpo monoclonal de reconhecida alta espécie-especificidade (clone B-18, marcador consagrado de Leishmania (Viannia) braziliensis), revelando a existência de dois tipos de serodemas entre as cepas estudadas. Cinco dos oito extratos solúveis de Leishmania foram submetidos à eletroforese em gel de amido de camada fina e subseqüentemente examinadas a fim de constatar a atividade das enzimas MPI, MDH, 6PGD, NH, NH-D, SOD, GPI e ES. Fundamentados nos estudos dos corridos eletroforéticos obtidos nos ensaios isoenzimáticos chegamos à conclusão que as cepas MHOM/AR/92 SE-1; SE-2; SE-4; SE-8 e SE-8-I são Leishmania (Viannia) braziliensis. É necessário analisar mais enzimas antes de enquadrá-los nos zymodema braziliensis

LEISHMANIASIS

Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes

Leishmaniose cutânea humana causada por Leishmania (Viannia) braziliensis na Província de Santiago del Estero, Argentina: identificação dos parasitas por anticorpos monoclonais e isoenzimas

C.A. Cuba Cuba^I; C.O. Torno^II; O. Ledesma^III; E. Visciarelli^II; S. Garcia^II; M.I. Prat^II; R. Costamagna^II; L. Barbieri^III; D.A. Evans^IV

^IUniversidade de Brasília, 70910-900, Brasília, D.F., Brazil

^IIUniversidad Nacional del Sur, Bahia Blanca, 8000, Argentina

^IIIHospital Independencia, Santiago del Estero, Argentina

^IVLondon School of Hygiene and Tropical Medicine, London, UK

^{Correspondence to} Correspondence to: C. A. CUBA CUBA

SUMMARY

Diagnostic and parasite characterization and identification studies were carried out in human patients with cutaneous leishmaniasis lesions in Santiago del Estero, Northern Province of Argentina. Diagnostic procedures were biopsies of lesions for smears and inoculations in hamster, needle aspirations of material from ulcers for "in vitro" cultures. Immunodiagnostic techniques applied were IFAT-IgG and Montenegro skin test. Primary isolation of eight stocks of leishmanial parasites was achieved from patients with active lesions. All stocks were biologically characterized by their behaviour in hamster, measurements of amastigote and promastigotes and growth "in vitro".

Eight stocks were characterized and identified at species level by their reactivity to a cross-panel of sub-genus and specie-specific Monoclonal Antibodies through an Indirect Immunofluorescence technique and a Dot-ELISA.

We conclude from the serodeme analysis of Argentina stocks that: stocks MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 and SE-36 are Leishmania (Viannia) braziliensis. Three Leishmania stocks (SE-1; SE-2 and SE-30) did not react with one highly specie-specific Monoclonal Antibody (Clone: B-18, Leishmania (Viannia) braziliensis marker) disclosing two serodeme group patterns.

Five out of eight soluble extracts of leishmanial promastigotes were electrophoresed on thin-layer starch gels and examined for the enzyme MPI, Mannose Phosphate Isomerase; MDH, Malate Dehydrogenase; 6PGD, 6 Phosphogluconate Dehydrogenase; NH, Nucleoside Hydrolase, 2-deoxyinosinc as substrate; SOD, Superoxide Dismutase; GPI, Glucose Phosphate Isomerase and ES, Esterase. From the isoenzyme studies we concluded that stocks: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8 and SE-8-I are isoenzymatically Leishmania (Viannia) braziliensis. We need to analyze more enzymes before assigning them to a braziliensis zymodeme.

Keywords: Cutaneous leishmaniasis; Leishmania (Viannia) braziliensis; Identification; Santiago del Estero, Argentina.

RESUMO

Estudos de diagnóstico, caracterização parasitária e identificação foram conduzidos em pacientes humanos com lesões cutâneas de leishmaniose na Província de Santiago del Estero, no Norte da Argentina. Os procedimentos de diagnóstico foram: biópsias de lesões para utilização em esfregaços e inoculação em hamster; aspiração (com agulha) de úlceras, para cultura "in vitro". As técnicas imunodiagnósticas empregadas foram a IFAT-IgG e o teste intradérmico de Montenegro. Oito cepas de parasitas foram isoladas, sendo estas obtidas de pacientes com lesões ativas. Todas as cepas foram inicialmente caracterizadas biologicamente por seu comportamento na infecção experimental do hamster, mensuração dos amastigotas e promastigotas e crescimento "in vitro".

As mesmas oito cepas foram logo identificadas e caracterizadas a nível de espécie, devido a sua reatividade frente a um painel de anticorpos monoclonais subgênero e espécie-específicos. Isso foi realizado utilizando o teste de Imunofluorescência Indireta (IFAT/MAbs) e de um procedimento de Dot-ELISA.

Nós concluímos a partir da análise de serodema dos isolados argentinos que: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 e SE-36 são Leishmania (Viannia) braziliensis. Entretanto, três dos isolados de Leishmania (SE-1; SE-2 e SE-30) não foram reconhecidos quando testados com um anticorpo monoclonal de reconhecida alta espécie-especificidade (clone B-18, marcador consagrado de Leishmania (Viannia) braziliensis), revelando a existência de dois tipos de serodemas entre as cepas estudadas.

Cinco dos oito extratos solúveis de Leishmania foram submetidos à eletroforese em gel de amido de camada fina e subseqüentemente examinadas a fim de constatar a atividade das enzimas MPI, MDH, 6PGD, NH, NH-D, SOD, GPI e ES. Fundamentados nos estudos dos corridos eletroforéticos obtidos nos ensaios isoenzimáticos chegamos à conclusão que as cepas MHOM/AR/92 SE-1; SE-2; SE-4; SE-8 e SE-8-I são Leishmania (Viannia) braziliensis. É necessário analisar mais enzimas antes de enquadrá-los nos zymodema braziliensis.

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10. GRIMALDI Jr., G.; TESH, R.B. & McMAHON-PRATT, D. - A review of the geographical distribution and epidemiology of leishmaniasis in the New World. Amer. J. trop. Med. Hyg., 4: 687-725, 1989.

Recebido para publicação em 15/04/1996

Aceito para publicação em 20/12/1996

1. ARANA, M.; EVANS, D.A.; ZOLESSI, A.; LLANOS-CUENTAS, A. & AREVALO, J. - Biochemical characterization of Leishmania (Viannia) braziliensis and Leishmania (Viannia) peruviana by isoenzyme electrophoresis. Trans. roy. Soc. trop. Med. Hyg., 84: 526-529, 1990.
2. BERNASCONI, V. - Consideraciones sobre la 5Ş Reunión de la Sociedad de Patologia Regional del Norte (Jujuy) M.E.P.R.A. Jujuy, Imprenta de la Universidad de Buenos Aires, 1929. p. 561-573.
3. CUBA CUBA, C.A.; MARSDEN, P.D.; BARRETO, A.C. et al. - Parasitological and immunological diagnosis of American mucocutaneous leishmaniasis. Bull. pan-Amer.Hlth. Org., 15: 249-259, 1981.
4. CUBA CUBA, C.A.; MARSDEN, P.D.; BARRETO, A.C.; JONES, T.C. & RICHARDS, R. - Use of different concentrations of leishmanial antigen in skin testing to evaluate delayed hypersensitivity in American mucocutaneous leishmaniasis. Rev. Soc. bras. Mod. trop., 18: 231-236, 1985.
5. CUBA CUBA, C.A.; MILES, M.A.; VEXENAT, A. et al. - A locus of mucocutaneous leishmaniasis in Tres Braços, Bahia, Brazil: characterization and identification of Leishmania stocks isolated from man and dogs. Trans. roy. Soc. trop. Med. Hyg., 79: 500-507, 1985.
6. DESJEUX, P. - Information on the epidemiology and control of the leishmaniases by country or territory. WHO/LEISH/91.30, p. 47 (Mimeographed document. 1991).
7. EVANS, D.A.; LANHAM, S.M.; BALDWIN, C.I. & PETERS, W. - The isolation and isoenzyme characterization of Leishmania braziliensis subsp. from patients with cutaneous leishmaniasis acquired in Belize. Trans. roy. Soc. trop. Med. Hyg., 78: 35-42, 1984.
8. FALCOFF, E.; TARANTO, N.J.; REMONDEGUI,C.E. et al. - Clinical healing of antimony-resistant cutaneous or mucocutaneous leishmaniasis following the combined administration of interferon-gamma and pentavalent antimonial compounds. Trans. roy. Soc. trop. Med. Hyg., 88: 95-97, 1994.
9. GODFREY, D.G. & KILGOUR, V. - Enzyme electrophoresis in characterizing the causative organism of Ganibian trypanosomiasis. Trans. roy. Soc. trop. Med. Hyg., 70: 219-224, 1976.
11. MARSDEN, P.D. - Endemic leishmaniasis in Brazil: future implication. Mem. Inst. Oswaldo Cruz, 89: 425-426, 1994.
12. McMAHON-PRATT, D.; JAFFE, C. & GRIMALDI Jr., G. - Application of monoclonal antibodies to the classification of Leishmania species. In: MOREL, C.M., ed. Genes and antigens of parasites. A laboratory manual. Rio de Janeiro UNDP/WORLD BANK/WHO-Special Program for Research and Training in Tropical Diseases-FIOCRUZ, 1984. p. 121-135.
13. MAZZA, S. - Leishmaniasis tegumentaria y visceral. M.E.P.R.A. Bol. Inst. Clin. quir. (B. Aires), 13: 1-24, 1926.
14. MAZZA, S. - Leishmaniasis cutanea en el caballo y nueva observacion de la misma en el perro. Bol. Inst. Clin. quir. (B. Aires), 13: 462-464, 1927.
15. PATEL, J.D.; JOSEPH, J.M. & FALKLER Jr., W. A. - Direct detection of Chlamydia trachomatis in clinical specimen by a dot-immunobinding technique using monoclonal antibody. J. immunol. Meth., 108: 279-287, 1988.
16. PETERS, W.; EVANS, D.A. & LANHAM, S.M. - Importance of parasite identification in cases of leishmaniasis. J. roy. Soc. Med., 76: 540-542, 1983.
17. PETERSON, N.E.; COSTA, M.A.F.; VEXENAT. A. et al. - Recovery of Leishmania (Viannia) braziliensis from inoculated hamsters. J. Parasit., 74: 512-514, 1988.
18. ROMAÑA, C. & ABALOS, J.W. - Distribution de flebótomos en la Argentina. An. Inst. Med. region. (Corrientes), 2: 293-302, 1949.
19. ROMAŃA, C.; NAJERA, L.; CONEJOS, M. & ABALOS, J.W. - Leishmaniasis tegumentaria en perros de Tucumán. Foco doméstico de leishmaniasis. An. Inst. Med. region. (Corrientes), 2: 282-292, 1949.
20. SALOMON, D.; TRAVI, B.L. & SEGURA, E.L. - Note on sandflies associated with a tegumenlary leishmaniasis focus in Salta, Argentina 1988. Rev. Inst. Med. trop. S. Paulo, 37: 91-92, 1995.
21. TRAVI, B.L.; REY-LADINO, J. & SARAVIA, N.G. - Behavior of Leishmania braziliensis s.l. in golden hamsters: evolution of the infection under different experimental conditions. J. Parasit., 74: 1059-1062, 1988.
22. WORLD HEALTH ORGANIZATION - Control of the leishmaniasis. Wld. Hlth. Org. techn. Rep. Ser., (793), 1990.

Correspondence to:

C. A. CUBA CUBA

Publication Dates

Publication in this collection
22 Sept 2006
Date of issue
Dec 1996

History

Received
15 Apr 1996
Accepted
20 Dec 1996

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. ARANA, M.; EVANS, D.A.; ZOLESSI, A.; LLANOS-CUENTAS, A. & AREVALO, J. - Biochemical characterization of Leishmania (Viannia) braziliensis and Leishmania (Viannia) peruviana by isoenzyme electrophoresis. Trans. roy. Soc. trop. Med. Hyg., 84: 526-529, 1990.

[2] 2. BERNASCONI, V. - Consideraciones sobre la 5Ş Reunión de la Sociedad de Patologia Regional del Norte (Jujuy) M.E.P.R.A. Jujuy, Imprenta de la Universidad de Buenos Aires, 1929. p. 561-573.

[3] 3. CUBA CUBA, C.A.; MARSDEN, P.D.; BARRETO, A.C. et al. - Parasitological and immunological diagnosis of American mucocutaneous leishmaniasis. Bull. pan-Amer.Hlth. Org., 15: 249-259, 1981.

[4] 4. CUBA CUBA, C.A.; MARSDEN, P.D.; BARRETO, A.C.; JONES, T.C. & RICHARDS, R. - Use of different concentrations of leishmanial antigen in skin testing to evaluate delayed hypersensitivity in American mucocutaneous leishmaniasis. Rev. Soc. bras. Mod. trop., 18: 231-236, 1985.

[5] 5. CUBA CUBA, C.A.; MILES, M.A.; VEXENAT, A. et al. - A locus of mucocutaneous leishmaniasis in Tres Braços, Bahia, Brazil: characterization and identification of Leishmania stocks isolated from man and dogs. Trans. roy. Soc. trop. Med. Hyg., 79: 500-507, 1985.

[6] 6. DESJEUX, P. - Information on the epidemiology and control of the leishmaniases by country or territory. WHO/LEISH/91.30, p. 47 (Mimeographed document. 1991).

[7] 7. EVANS, D.A.; LANHAM, S.M.; BALDWIN, C.I. & PETERS, W. - The isolation and isoenzyme characterization of Leishmania braziliensis subsp. from patients with cutaneous leishmaniasis acquired in Belize. Trans. roy. Soc. trop. Med. Hyg., 78: 35-42, 1984.

[8] 8. FALCOFF, E.; TARANTO, N.J.; REMONDEGUI,C.E. et al. - Clinical healing of antimony-resistant cutaneous or mucocutaneous leishmaniasis following the combined administration of interferon-gamma and pentavalent antimonial compounds. Trans. roy. Soc. trop. Med. Hyg., 88: 95-97, 1994.

[9] 9. GODFREY, D.G. & KILGOUR, V. - Enzyme electrophoresis in characterizing the causative organism of Ganibian trypanosomiasis. Trans. roy. Soc. trop. Med. Hyg., 70: 219-224, 1976.

[10] 11. MARSDEN, P.D. - Endemic leishmaniasis in Brazil: future implication. Mem. Inst. Oswaldo Cruz, 89: 425-426, 1994.

[11] 12. McMAHON-PRATT, D.; JAFFE, C. & GRIMALDI Jr., G. - Application of monoclonal antibodies to the classification of Leishmania species. In: MOREL, C.M., ed. Genes and antigens of parasites. A laboratory manual. Rio de Janeiro UNDP/WORLD BANK/WHO-Special Program for Research and Training in Tropical Diseases-FIOCRUZ, 1984. p. 121-135.

[12] 13. MAZZA, S. - Leishmaniasis tegumentaria y visceral. M.E.P.R.A. Bol. Inst. Clin. quir. (B. Aires), 13: 1-24, 1926.

[13] 14. MAZZA, S. - Leishmaniasis cutanea en el caballo y nueva observacion de la misma en el perro. Bol. Inst. Clin. quir. (B. Aires), 13: 462-464, 1927.

[14] 15. PATEL, J.D.; JOSEPH, J.M. & FALKLER Jr., W. A. - Direct detection of Chlamydia trachomatis in clinical specimen by a dot-immunobinding technique using monoclonal antibody. J. immunol. Meth., 108: 279-287, 1988.

[15] 16. PETERS, W.; EVANS, D.A. & LANHAM, S.M. - Importance of parasite identification in cases of leishmaniasis. J. roy. Soc. Med., 76: 540-542, 1983.

[16] 17. PETERSON, N.E.; COSTA, M.A.F.; VEXENAT. A. et al. - Recovery of Leishmania (Viannia) braziliensis from inoculated hamsters. J. Parasit., 74: 512-514, 1988.

[17] 18. ROMAÑA, C. & ABALOS, J.W. - Distribution de flebótomos en la Argentina. An. Inst. Med. region. (Corrientes), 2: 293-302, 1949.

[18] 19. ROMAŃA, C.; NAJERA, L.; CONEJOS, M. & ABALOS, J.W. - Leishmaniasis tegumentaria en perros de Tucumán. Foco doméstico de leishmaniasis. An. Inst. Med. region. (Corrientes), 2: 282-292, 1949.

[19] 20. SALOMON, D.; TRAVI, B.L. & SEGURA, E.L. - Note on sandflies associated with a tegumenlary leishmaniasis focus in Salta, Argentina 1988. Rev. Inst. Med. trop. S. Paulo, 37: 91-92, 1995.

[20] 21. TRAVI, B.L.; REY-LADINO, J. & SARAVIA, N.G. - Behavior of Leishmania braziliensis s.l. in golden hamsters: evolution of the infection under different experimental conditions. J. Parasit., 74: 1059-1062, 1988.

[21] 22. WORLD HEALTH ORGANIZATION - Control of the leishmaniasis. Wld. Hlth. Org. techn. Rep. Ser., (793), 1990.

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Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes

Leishmaniose cutânea humana causada por Leishmania (Viannia) braziliensis na Província de Santiago del Estero, Argentina: identificação dos parasitas por anticorpos monoclonais e isoenzimas

Abstracts

Correspondence to:

Publication Dates

History