Comparación de métodos convencionales y moleculares para la detección de giardia lamblia en heces humanas

Abstract

Abstract

The protozoa of the genus Giardia represent one of the most common human parasites in the world and are among the main causes of gastrointestinal infections and diarrheal diseases in humans. Parasite detection is generally based on concentration and conventional microscopy methods, but these techniques have limitations due to their low sensitivity and specificity in diagnosis. In an attempt to improve the diagnostic methods, molecular techniques are emerging as a promising alternative. In this study 88 stool samples from patients of a company that provides health services (ASSBASALUD) in the city of Manizales (Caldas) were analyzed. In order to detect Giardia lamblia in stool, three different methods were compared using the positivity percent: conventional methods for concentration of the sample and microscopic observation, analysis by immunoassay (indirect ELISA) and finally the amplification of two nuclear gene sequences by PCR. Three positive samples were obtained by concentration and conventional microscopy, two by immunoassay and 26 by molecular techniques. The study suggests that routine diagnostic tests based on conventional microscopy and immunoassay have lower detection rate of this parasite and that this deficiency can be compensated by means of the implementation of molecular diagnostic methods based on PCR, as a complementary strategy to support the diagnosis of this protozoan.

Keywords:

Giardia lamblia

inmunoensayo

PCR

Giardia lamblia

immunoassay|

PCR

References

 Adam, R.D. (2001). Biology of Giardia lamblia. Clinical Microbiol. Reviews, 14, 447-475.

 Barrientos, P.D., Torrico, M.C. y Suárez, E. (2008). Detección de Cryptosporidium spp. y Giardia lamblia en niños inmunodeprimidos del Hospital del Niño Manuel Ascencio Villarroel de Cochabamba en
Agosto de 2007. GMB, 31(1), 45-49.

 Beaver, P.C., Jung, R.C, y Cupp, E.W. (1984). Clinical Parasitology (9th ed.). Philadelphia: Lea and Febiger. p. 52-56.

 Braga, A.M. y Catapani, W.R. (2005). Enzyme-linked immunosorbent assay (ELISA) immunoassaying versus microscopy: advantages and drawbacks for diagnosing giardiasis. Sao Paulo Med. J., 123(6), 282- 285.

 Cacciò, S.M., De Giacomo, M., Aulicino, F.A. y Pozio, E. (2003). Giardia cysts in wastewater treatment plants in Italy. Appl. Environ. Microbiol., 69(6), 3393-3398.

 Carmona, J., Uscátegui , R.M. y Correa, A.M. (2009). Parasitosis intestinal en niños de zonas palúdicas de Antioquia (Colombia). Iatreia, 22(1), 27-46.

 Chaves, M.P., Fernández, J.A., Ospina, I., López, M.C., Moncada, L. y Reyes, P. (2007). Tendencia de la prevalencia y factores asociados a la infección por Giardia duodenalis en escolares y preescolares de una zona rural de Cundinamarca. Biomédica, 27, 345-351.

 Corinne, F.L., Amar, P.H. y McLauchlin, J. (2003). Detection and genotyping by real-time PCR/RFLP analyses of Giardia duodenalis from human faeces. J Med Microbiol., 52, 681-683.

 Fernández, P.S. y Díaz, P.S. (2003). Pruebas diagnósticas. Cad Aten Primaria, 10, 120-124.

 Gonçalves, E.M.N., Araújo, R.S., Orban, M., Matté, G.R., Matté, M.H. y Corbett, C.E.P. (2008). Protocol for DNA extraction of Cryptosporidium spp. oocysts in fecal samples. Rev. Inst. Med. trop. S. Paulo, 50(3), 165-167.

 González de la Rosa, J.B., Barbadillo Izquierdo, F., Merino Arribas, J.M. y Sánchez, J. (1999). Aparato Digestivo. Parasitosis intestinales. Protocolo diagnóstico-terapéutico. Bol Pediatr, 39, 106- 111.

 Inpankaew, T., Traub, R., Thompson, R.C. y Sukthana, Y. (2007). Canine parasitic zoonoses in Bangkok temples. Southeast Asian J Trop Med Public Health, 38(2), 247-55.

 Izquierdo, A., Mendoza, D., Sarria, C.A. y Álvarez, G. (2006). Prevalencia de Parasitosis Intestinal en niños de nivel primario de la Institución Educativa Juan María Rejas de la localidad Tacneña de Pachía, Perú. Revista Ciencias.com. Recuperado de http://www.ilustrados.com/tema/8809/Prevalencia-ParasitosisIntestinal-ninos-nivel-primario.html

 Johnston, A.R., Gillespie, T.R., Rwego, I.B., McLachlan, T.L., Kent,
A.D. y Goldberg, T.L. (2010). Molecular epidemiology of crossspecies Giardia duodenalis transmission in western Uganda. PLoS Negl Trop Dis., 4(5), e683. doi: 10.1371/journal.pntd.0000683.

 Lora-Suárez, F., Marín-Vásquez, C., Loango, N., Gallego, M., Torres, E., González, M., Castaño-Osorio, J. y Gómez-Marín J. (2002). Giardiasis in children living in post-earthquake camps from Armenia (Colombia). BMC Public Health, 2. Recuperado de http://download.springer.com/static/pdf/306/art%253A10.1186%2 52F1471-2458-2- 5.pdf?auth66=1385319461_0e96cf4c940760e2af3743cb73a649f3 &ext=.pdf

 Medina, L., García, M.G., Galván, A.L. y Botero, J. (2009). Prevalencia de parásitos intestinales en niños que asisten al Templo Comedor Sagrado Corazón Teresa Benedicta de la Cruz, del barrio Vallejuelos, Medellín, 2007. Iatreia, 22(3), 227-234.

 Miller, K.M. y Sterling, C.R. (2007). Sensitivity of Nested PCR in the detection of low numbers of Giardia lamblia cysts. Applied and environmental microbiology, 73(18), 5949-5950.

 Molina, N.B. (2009). Epidemiología molecular de Giardia lamblia en comunidades urbanas y rurales de Buenos Aires y Mendoza, Argentina. (Tesis de Magíster en Ciencias del Laboratorio Clínico). Facultad de Ciencias Exactas. Universidad Nacional de La Plata, Argentina.

 Molina, N.B., Polverino, P., Minvielle, M.C., Apezteguía, M., Aguilar, M. y Basualdo, J.A. (2006). Comparación de métodos de lisis y extracción de ADN de trofozoítos de Giardia lamblia. Parasitol Latinoam, 61, 133-137.

 Monis, P.T., Andrews, R.H., Mayrhofer, G. y Ey, P.L. (1999). Molecular Systematics of the Parasitic Protozoan Giardia intestinalis. Mol Biol Evol., 16(9), 1135-1144.

 Núñez, F.A. (2004). Estudio de factores asociados con la reinfección por Giardia lamblia en niños de círculos infantiles. (Tesis de Doctorado). Departamento de Parasitología, Subdirección de Parasitología, Instituto de Medicina Tropical “Pedro Kourí”. Cuba.

 Paulino, R.C. (2005). Detección Molecular de Giardia sp. en muestras fecales y agua: Extracción de ADN genómico, PCR y RFLP. (Tesis de Postgrado). Universidad Federal de Paraná, Brasil.

 Robertson, L.J., Forberg, T., Hermansen, L., Hamnes, I.S. y Gjerde, B. (2007). Giardia duodenalis cysts isolated from wild moose and reindeer in norway: genetic characterization by PCR-RFLP and sequence analysis at two genes. J Wildl Dis., 43(4), 576-585.

 Sanguinetti, C., Dias, N. y Simpson, A. (1994). Rapid silver staining and recovery of PCR products separates on polycrylamide gels. Biotechniques, 17, 914-919.

 Sulaiman, I.M., Fayer, R., Bern, C., Gilman, R.H., Trout, J.M., Schantz, P., Das, P., Lal, A.A. y Xiao, L. (2003). Triosephosphate Isomerase Gene Characterization and Potential Zoonotic Transmission of Giardia duodenalis. Enfermedades Infecciosas Emergentes, 9(11), 1444-1452.

 Téllez, A., Winiecka-Krusnell, J., Paniagua, M. y Linder, E. (2003).Antibodies in mother’s milk protect children against giardiasis. Scand J Infect Dis, 35(5), 322-325.

 Winkworth, C.L., Learmonth, J.J., Matthaei, C.D. y Townsend, C.R. (2008). Molecular characterization of Giardia isolates from calves and humans in a region in which dairy farming has recently intensified. Appl Environ Microbiol, 4(16), 5100-5105. doi: 10.1128/AEM.

 World Health Organization. (2002). Guidelines for drinking water quality. Addendum: Microbial agents in drinking water (2nd ed.). Geneva, Switzerland: WHO.

 Yason, J.A. y Rivera, W.L. (2007). Genotyping of Giardia duodenalis isolates among residents of slum area in Manila, Philippines. Parasitol. Res., 101, 681-687.